22-Kilodalton (kDa) protein cDNA clones were isolated from a rat prostatic library. Nucleotide sequence analysis revealed three different cDNA sequences encoding two somewhat different open reading frames of 176 amino acids. The N-terminal 24 amino acids of these sequences show the typical characteristics of signal peptides of secretory proteins. The C-terminal end of the derived protein sequences displays sequence similarity to a number of cysteine proteinase inhibitors, called cystatins, suggesting a common physiological function. Upon Northern blotting with a labeled cDNA fragment, three different 22-kDa protein mRNAs, i.e. 950 nucleotides (nt), 920 nt and 860 nt, could be detected in the rat ventral prostate and the lacrymal gland. In both tissues these messengers were regulated by androgens showing the most rapid androgen response for the 950 nt mRNA form. Administration of cycloheximide nearly completely abolished the observed androgen effect suggesting that a short-living protein is required for the full induction of the 22-kDa protein genes. Hybridization experiments with specific oligonucleotides which distinguish between the mRNAs encoding both 22-kDa protein variants indicate that one protein form is less androgen dependent in the ventral prostate and not expressed in the lacrymal gland.