Past studies have reported the appearance of cells sharing phenotypic characteristics of gonadotropes and GH cells. During diestrus and early proestrus, a subset of somatotropes (40-60%) expressed both GH antigens and gonadotropin (LH-beta, LHbeta, or FSH-beta) messenger RNAs (mRNAs) or GnRH receptors. More recently, we reported that subsets of gonadotropes identified by LHbeta or FSHbeta antigens expressed GH- releasing hormone (GHRH) binding sites. The present studies were designed to learn if these putative multipotential cells also expressed GH mRNA. Biotinylated sense and antisense oligonucleotide probes were developed and cytochemical in situ hybridization tests were optimized for the detection of GH mRNA with GH, LHbeta, and FSHbeta antigens. RNase protection assays were developed with a complementary RNA probe that detected a 380-bp region at the 5' end of the GH mRNA. Both the in situ hybridization and RNase protection assays detected changes in expression of GH mRNA during the estrous cycle with the lowest expression occurring during metestrus and peak expression occurring on the morning of proestrus. Cell counts confirmed the results of the RNase protection assays showing that increases in mRNA levels seen from metestrus to proestrus reflected increased percentages of GH mRNA-bearing cells. In addition, densitometric analyses demonstrated that the higher GH mRNA levels assayed from diestrus to proestrus reflected increased area and density of label per cell. Both types of assays showed sex differences in expression of GH mRNA; male rat cell populations had higher values than female rats in metestrus, diestrus, or estrus. However, percentages of GH cells in male rats were equal to those from proestrous female rats and levels of GH mRNA were lower in male rats than proestrous females. Dual labeling experiments showed that, in male rats and diestrous, proestrous, or estrous females, GH mRNA was expressed in over 70% of GH cells. Expression of GH mRNA was also found in 50-57% of cells with LHbeta or FSHbeta antigens in the same groups. The lowest expression was seen in the metestrous groups (30-40% of GH cells or gonadotropes expressed GH mRNA). Expression of GH mRNA was first increased from metestrus to diestrous largely in GH cells, and slightly in cells with LHbeta antigens. Further increases were seen in GH and LH cells by the morning of proestrus. In contrast, FSH gonadotropes did not show an increased expression of GH mRNA until the morning of proestrus (reaching the same peak reached by LH cells). These data confirm the working hypothesis that a multihormonal cell type develops during diestrus to support both the somatotrope and gonadotrope populations. Collectively, our studies suggest that this multihormonal cell may function to help support the regulatory functions of the gonadotrope during the periovulatory period. In addition, the appearance of significant levels of expression of GH mRNA by male rat gonadotropes suggests that this multihormonal cell may play a role in regulation of the male reproductive system as well.