We examined the biological properties of two infectious clones of a simian immunodeficiency virus, SIVMND, which were designated as pMD121 and pMD122. Upon transfection into CD4-negative cells, pMD122 generated virions much less efficiently than pMD121. Likewise, the growth kinetics in CD4-positive cells of virus derived from pMD122 were remarkably delayed relative to those of virus from pMD121. The cytocidal activity of the MD122 virus was also low. A series of recombinant clones were constructed from pMD121 and pMD122 to determine the sequence responsible for the low virulence of the MD122 virus. The genetic determinant in pMD122 responsible for its properties was mapped to within a region (316 base pairs) encompassing the tat, rev, and env coding sequences. Sequence analysis revealed that the two clones differed by only one nucleotide in this region. A nucleotide substitution G (pMD121) to T (pMD122) altered an arginine codon to a serine codon in the first tat coding exon. Transient transfection experiments showed that the tat activity of pMD122 was about twofold less than that of pMD121. These findings indicate that small differences in tat activity can have a dramatic effect on the biological behavior of SIVMND.