We have purified and characterized a cathepsin L-specific inhibitor (psoriastatin) from psoriatic epidermis. This inhibitor is distinct from cystatins, members of the well known cysteine proteinase inhibitor family, in terms of molecular weight and inhibition spectrum. In the present study we report on the cloning of two types of psoriastatin cDNAs. Approximately 4 mg of poly(A) + RNA were obtained from psoriatic skin through shave biopsy. A psoriasis cDNA library was constructed using the λZAP Express cDNA Synthesis Kit (Stratagene). cDNAs of around 2,000 bp were inserted into the ZAP Express vector and packaged with the Gigapack II Gold packaging extract (Stratagene). The library was screened with a cDNA fragment obtained by PCR using primers that were based on the sequence of a tryptic peptide. After the tertiary screening, 25 positive clones were isolated and analyzed. None of these clones contained 5 end sequences, however. Full length clones were obtained using the 5 RACE system (Gibco). Restriction enzyme analysis of these clones using Pst I digestion demonstrated two distinct groups of psoriastatin cDNAs. The sequence of psoriastatin type I was found to be almost identical to that of squamous cell carcinoma antigen (SCC-A). The msequence of psoriastatin type II was considerably different from type I in both the coding and the 3 -untranslated regions. Alignment analysis of the predicted proteins using the GCG program revealed that type I and type II were 98% and 91% homolous with SCC-A, respectively. Both types were subcloned into pGEX-4T-2 vector and expressed as GST-fusion proteins. Only psoriastatin type I showed inhibitor activity against cathepsin L, demonstrating a functional difference between type I and type II psoriastatin.