Antilymphocyte antibodies (ALA) in rheumatoid arthritis (RA) have increased reactivity with phytohemagglutinin (PHA) activated lymphoblasts which are known to have increased expression of Ia antigen. The present experiments suggest that part of this reactivity represents an Ia specificity of ALA. Fifteen of 18 RA sera tested were able to inhibit the binding of monoclonal anti-Ia antibodies as measured by a rosette method. RA sera did not inhibit the binding of other monoclonal antibodies: anti-OKT3, anti-OKT4, and anti-OKT8. The ability of RA sera to inhibit the binding of anti-Ia antibody was eliminated after absorption of the RA sera with an Ia positive human cell line (B35M) but not by an Ia negative line (MOLT4). Blocking of anti-Ia binding was greater in the IgG fraction of the RA sera but also occurred in the IgM fraction. Experiments including ultracentrifugation, pepsin digestion of RA sera, and preincubation of lymphoblasts with aggregated IgG demonstrated that Fc binding by RA sera was not a factor. Both monoclonal anti-Ia and anti-Ia heteroantiserum also had increased reactivity with lymphoblast target cells. Pepsin digested Fab fragments of the anti-Ia heteroantiserum were able to block the activity of cytotoxic RA serum. However, ALA cytotoxic to lymphoblasts did not correlate with anti-Ia by rosette method. Ia-specific ALA by rosette method was associated with donor variability but did not appear to be HLA-DR restricted. Ia-specific ALA did correlate with disease activity. These data suggest that anti-Ia activity is present in RA sera and may play an immunoregulatory role in this disease.