Monoclonal antibodies (MAbs) against the major gag protein (p34) of a retrovirus produced in a human lymphoblastoid cell line (HLB) were prepared for the characterization of this virus. Immunoelectron microscopy showed that one of these MAbs, MAb 14H10, immunoreacted specifically with the core protein of this virus. p34 and its partially degraded products were recovered in a one-step manipulation from the crude extract of HLB cells by immunoaffinity chromatography using MAb 14H10. p34 was highly purified by polyacrylamide gel electrophoresis, and the amino acid sequence of 27 amino acid residues in the N terminal region of the purified p34 was determined by a gas-phase microsequenator. The fact that the sequence begins with Pro-Val- confirmed that p34 was the major gag protein of this virus. Comparing the N-terminal sequence (27 amino acid residues) of p34 with the known counterpart sequences of major gag proteins of other retroviruses, no retroviruses highly homologous in the N-terminal region were found. The N-terminal sequence (Pro-Val-) of p34 and Mg(2+) preference of the reverse transcriptase of this virus suggested that this virus belonged to a retrovirus other than mammalian type C retroviruses whose prototype is Moloney murine leukemia virus having the N-terminal sequence (Pro-Leu-Arg-) of the major gag protein and Mn(2+) preference of the reverse transcriptase.