The cattle protozoan parasite Tritrichomonas foetus has multiple forms of cysteine proteinases. To investigate their diversity, PCR and reverse transcriptase PCR were used to isolate genomic DNA and cDNA fragments, respectively, encoding different cysteine proteinases. Seven genes have been identified, TFCP3-6 from amplification of genomic DNA and TFCP7-9 from amplification of cDNA. Comparison of the predicted amino acid sequences indicates that the T. foetus enzymes are cathepsin-L-like rather than cathepsin-B-like in structure. However, there is considerable diversity among the proteinases. TFCP7 and TFCP8 are most similar to one another (78% identity), while TFCP3 and TFCP9 are the least closely related (30% identity). All but one of the genes are single-copy, the exception being TFCP3, which was present in multiple copies in one of the three isolates examined. Single transcripts were detected for each of the seven genes. TFCP8 was expressed at the highest levels, while transcripts for TFCP4 were only just detectable. In T. foetus F2, the strain from which the genomic DNA and mRNA were isolated, transcripts of the five other genes were present at intermediate levels. When two other isolates were compared with F2, differences in the expression of individual genes were apparent, with either one or two of them not expressed. In spite of these differences the major cysteine proteinases detected in the three isolates using substrate-SDS-PAGE appeared identical. The data show that the multiplicity of cysteine proteinases in T. foetus is due, in part at least, to the presence of multiple genes and that some of the genes encode cysteine proteinases which are not among the high-activity enzymes detected previously.