In this report we describe the discovery of Eppin (Epididymal protease inhibitor), a gene on human chromosome 20 expressing three mRNAs encoding two isoforms of a cystine-rich protein containing both Kunitz-type and WAP-type four disuffide core protease inhibitor consensus sequences. Analysis of Eppin's genomic sequence from chromosome 20q12-13.2 predicts the existence of all three splice variants of Eppin and that all the exons conform to the AG/GT splicing rule. The presence of single bands on a Southern blot of human genomic DNA suggests that Eppin is a single copy gene. TATA box transcription initiation sites are present for both of the different Eppin 5' UTRs and examination of the promoter region 1800 bp upstream of the start codon revealed a number of putative transcription enhancer binding sites typical of genes expressed in the epididymis or testis. Northern blot and tissue specific PCR data indicate Eppin-1 is expressed only in the testis and epididymis; Eppin-2 is expressed only in the epididymis and Eppin-3 only in the testis. Antiserum prepared against recombinant EPPIN recognizes several strong bands on Western blots of human epididymal extracts from the caput and corpus regions. Immunohistochemistry indicates a strong pattern of expression by the ciliated cells of the efferent ducts and strong staining of ejaculated spermatozoa. Eppin represents the first member of a family of protease inhibitors characterized by dual inhibitor consensus sequences, both WAP-type and Kunitz-type consensus sequences. A second family member is predicted to exist on chromosome 20 approximately 4 kb downstream from Eppin's exon I, which has two WAP-type sequences and one Kumtz-type consensus sequence.