HIV-1 replicates more efficiently in cultured IL-2-dependent CD4 T cells expressing V beta 12 T cell receptors (TCRs) rather than other TCRs (Laurence et al., 1992). A viral reservoir is frequently established in V beta 12 T cells in HIV-1-infected patients. Here we show that cytomegalovirus (CMV) is responsible for V beta 12-selective HIV-1 replication that is indistinguishable from the effect of known superantigens (SAGs). This effect is dependent on direct contact of T cells with CMV-infected monocytes. CMV infection, but not ie1 or ie2 transfection, reproduces this effect in a monocytoid cell line (U937). In HIV-infected patients, the presence of CMV antibodies correlates with an HIV-1 viral load preferentially skewed to the V beta 12 subset. Together, these data suggest that a CMV gene product is responsible for a SAG-driven V beta 12-selective HIV-1 reservoir in vivo.