Antibodies to U1 ribonucleoproteins produced by peripheral blood mononuclear cells from patients with connective tissue diseases were measured by a sensitive enzyme-linked immunosorbent assay and by immunoblotting. Mononuclear cells from patients with high-titer serum antibodies spontaneously started secreting IgG anti-U1 RNP antibodies on the second day after culture. The amount of anti-U1 RNP in the culture supernatants reached maximum level on day 8. The most effective production of anti-U1 RNP by B cells was observed when they were cultured in the presence of T cells and adherent cells. Mononuclear cells from patients without anti-U1 RNP antibodies or from normal subjects did not produce a measurable amount of anti-U1 RNP. Treatment of mononuclear cells by cycloheximide resulted in complete inhibition of anti-U1 RNP secretion, which indicates that antibody in the culture supernatants reflects the active biologic phenomenon in vitro. The methods described should be useful in the study of the cellular mechanisms involved in antinuclear antibody production of connective tissue diseases.