The biological activity of an anti-Sindbis monoclonal antibody (MCAB 49) has been explored. The antibody recognizes an epitope on the E2 glycoprotein of Sindbis virus and, in the presence of complement (C'), neutralizes virus infectivity. In the absence of C', reaction of the antibody with our laboratory strain of Sindbis, SB, increased the number of plaque-forming units (PFU) detected on baby hamster kidney (BHK) cells rather than neutralizing virus infectivity. The elevated titers of SB approached, but never exceeded, the number of virions calculated from the particle:PFU ratio, indicating that the additional PFU might have resulted from activation of normally noninfectious particles. The apparent activation could not be attributed to disaggregation of SB by MCAB 49 as shown by ultraviolet inactivation experiments with antibody-treated and untreated virus preparations. Fc receptors did not appear to be involved in the antibody-mediated activation. Fab' and F(ab')2 fragments of MCAB 49 also increased the number of observed PFU of SB. Control monoclonal antibodies of the same isotype, but specific for the tobacco etch virus capsid protein, were unable to compete for cellular binding sites with the SB/MCAB 49 complex. Rather, the SB/MCAB 49 complex appeared to utilize the same receptor(s) as SB in that SB and the SB/MCAB 49 complexes competed with each other for binding sites on BHK cells. Binding studies with 32P-labeled SB showed that a higher proportion of MCAB 49 activated virions than untreated virions associated with BHK cells. Moreover, activated virions were much less susceptible to elution. These results suggest that reaction which MCAB 49 may facilitate successful attachment of SB to its receptor, or receptors, on BHK cells.