To investigate whether HTLV‐I induces the development of complement‐dependent cytotoxic antibodies in humans, sera of asymptomatic HTLV‐I carriers and of patients suffering from tropical spastic paraparesis/HTLV‐I‐associated myelopathy (TSP/HAM) or adult T cell leukaemia (ATL) were used in a cytotoxicity assay against a panel of target cells. This panel included uninfected cell lines (CEM, Jurkat, Molt and H9), cell lines chronically infected with HTLV‐I (MT2, MT4, C91PL and HUT102), as well as lines H36 (H9 infected with HTLV‐I), H9‐IIIB (H9 infected with HIVIIIB) and H9‐MN (H9 infected with HIVMN). HTLV‐I+ sera induced lysis of H36 and of lines expressing HTLV‐I antigens in the presence of rabbit complement, but did not lyse cells in presence of human complement. The HTLV‐I+ sera also failed to lyse the HTLV‐I− lines and H9 cells, suggesting that lysis was specific for HTLV‐I. H36 cell lysis was prevented by IgG depletion of the sera and by dialysis of rabbit complement against EGTA or EDTA. Rabbit complement‐dependent cytotoxic antibodies were present in the sera of 14/14 HTLV‐I‐infected individuals; the highest titres were predominantly found in the sera of the TSP/HAM patients. Such antibodies were also detected in 5/5 individuals coinfected with HIV‐1 and HTLV‐I, although no cytotoxic antibody could be found against HIV‐infected cells. Vice versa, sera of HIV‐1‐infected individuals did not exert a lytic effect in the presence of complement (of human or rabbit origin) against HIV‐1‐ or HTLV‐I‐infected cells. Incubation of the sera of four HTLV‐I‐infected patients with HTLV‐I env‐specific synthetic peptides demonstrated that some of the complement‐dependent cytotoxic antibodies recognized epitopes located on gp46 between amino acids 190 and 209. There is no correlation of rabbit complement‐dependent cytotoxic HTLV‐I antibodies with the development of disease.