Experiments were performed seeking conditions for the optimum use of anti-T cell monoclonal antibodies in vivo in mice. Anti-L3T4 (CD4) and anti-Lyt2 (CD8) antibodies of different subclasses (IgG2b, IgG2a, and IgM) and species (rat or mouse) were used. The results showed that (i) intraperitoneal compared to intravenous administration of the different antibodies achieved the same serum levels whether in the presence or absence of the recipient's thymus; (ii) repeated treatment with a rat IgM anti-L3T4 or a rat IgG2b anti-Lyt2 antibody was followed by inability to detect serum levels of each antibody; (iii) in vivo treatment with these antibodies caused target cell lysis, target antigen masking without cell destruction, or target antigen modulation without cell destruction and the particular effect of a given antibody could not be predicted by its isotype or specificity; (iv) neither the C5 component of complement nor antibody-dependent cell-mediated cytotoxicity mediated the action of GK1.5 antibody in vivo; (v) dose-response curves of in vivo potency of a given antibody could not be predicted by in vitro assays; (vi) thymocytes were depleted by monoclonal antibody treatment by using 1000-fold more antibody than needed to deplete peripheral lymphocytes; (vii) the rate of return of target T cells after depletion in nonthymectomized mice depended on the dose of the antibody; and (viii) thymectomy prolonged the effect of most, but not all antibodies. In thymectomized mice, CD8+ cells remained almost undetectable for prolonged periods of time after depletion while CD4+ cells returned to approximately 30% of their original level and remained constant over time after initial complete depletion. These results provide useful data for the effective use of monoclonal anti-T cell antibodies in mice. They stress the difficulty of predicting the in vivo effects of monoclonal antibodies without actually testing them in vivo. They include new insights into mechanisms of action of monoclonal antibodies and the role of thymectomy in prolonging their effect. They describe the unrecognized ability of antibodies to deplete thymocytes.