The biological activity and immunoreactivity of serum prolactin (PRL) has been shown to fluctuate throughout the estrous cycle of the rat. Since the 24-kDa nonphosphorylated and phosphorylated isoforms from several species have also been shown to differ in their biological and immunoreactivities, we have investigated the possibility that the 24-kDa monomer isoform profile varied throughout the estrous cycle of the rat. The PRL isoform profile was assessed in homogenates of pituitaries and in short-term incubation media. Comparisons between homogenates, which always contained isoforms 1, 2, 3, and 3’ (numbered according to increasing acidity), and media showed nonproportional release of the isoforms at all stages. Of great interest were the release of isoform 1 (a nonphosphorylated form) only at estrus and the lack of release of isoform 3’ (a phosphorylated form) only in the afternoon of proestrus. This lack of release of 3’ was accompanied by a marked increase in the release of isoform 2 (the unmodified polypeptide). These results suggest a unique function for isoform 1 during estrus and a role for increased isoform 2 and absent isoform 3’ during the proestrus surge of PRL. Moreover, they suggest that fluctuations in the biological activity and immunoreactivity of serum PRL during the estrous cycle could be due, at least in part, to fluctuations in the isoform profile.