Profilin is a small actin-binding protein that is involved in diverse functions such as maintaining cell structure integrity, cell mobility, tumor cell metastasis, as well as growth factor signal transduction. In this paper, we describe the molecular cloning and characterization of a novel form of profilin, termed profilin-3, from rat kidney using a PCR-based procedure for isolating tissue-specific genes. The profilin-3 cDNA encoded 137 amino acids, and it shared extensive homology to profilin-1 and profilin-2 from mice and humans. More strikingly, the expression of profilin-3 was highly selective, and its mRNA was only found in the kidney and to a much lesser extent in the testis. The size of the mRNA for profilin-3 in the testis was 1.2 kb, while in kidney it was approximately 4.4–5 kb, suggesting the presence of tissue-specific transcription from different promoters. In addition, we also found that the expression of profilin-3 mRNA was significantly elevated in two types of renal diseases: diabetic nephropathy (db/db mice) and polycystic kidney diseases (cpk mice). Similar to profilin-1 and profilin-2, profilin-3 was localized in the cytoplasmic domain. Furthermore, it was capable of interacting with actin and poly- L-proline in an in vitro assay as well as in transfected cells. These results strongly suggest that, contrary to the ubiquitous presence of profilin-1 and profilin-2, there is a tissue-specific form of these cytoskeleton-regulatory proteins and that the kidney/testes-specific profilin-3 may play a unique role in renal and/or reproductive functions.