A provirus clone of simian T-cell leukemia virus isolated from a pigtailed monkey (PT-STLV), which is 90% homologous to HTLV-I, was shown to be biologically active in transfection assay. In transfected cells, gp61env, Pr55gag, and the mature gag proteins p24, p21, and p15 were detected, and type C particles were produced. The virus could be transmitted from the transfectants to recipient cells by cocultivation. In this biologically active provirus clone, a coding frame, possibly for protease, was identified between the gag and pol genes. The corrected sequence of the protease region of HTLV-I was also found to have a single open reading frame overlapping the gag and pol genes, although it has an amber codon in the middle of the frame. Thus, a single coding frame, which is different from those of gag and pol, is common to proteases of the HTLV family including HTLV-I.