Five classes of HIV-2 (isolate NIH-Z) Env polypeptides were produced in bacteria, purified, and tested in an immunoblot (Western blot) assay reactivity to antibodies in human sera. Only the proteins expressed from two regions (antigen pMZ996, residues 537-707; antigen pMZ945, residues 214-344) were found to be reactive. The pMZ996 antigen was found to react strongly with all (5/5) HIV-2 sera tested, while the pMZ945 antigen reacted weakly with 2 out of the 5 sera. Neither HIV-2 antigen reacted to sera from HIV-1-infected individuals. The pMZ996 antigen thus has potential usefulness in the development of immunological assays for distinguishing HIV-1 and HIV-2 infections. The levels of HIV-1 RNAs in various infected cells were determined by RNA gel blot (Northern blot) hybridization and correlated with levels of proteins observed in immunoprecipitation and immunoblot (Western blot) assays. HIV-1 isolate HTLV-IIIB-infected H9 cells (H9 is a T cell tumor line) that had been passaged for over one year were found to express much lower levels of viral RNAs than cells that were freshly cultured from frozen stocks. This was consistent with the finding that viral structural protein expression was lower in high-passage cells. The levels of viral expression in HIV-1-infected macrophages were found to be lower than that of high-passage infected H9 cells and was much less than that of freshly cultured infected H9 cells.