Abstract The effect of modification of amino groups on the binding activity of various antihapten antibodies was studied. The antibodies were prepared against positively charged, negatively charged, and neutral haptens. The lysyl residues in the antibody preparations were modified to an extent of 65–70 per cent with N-carboxy- DL-alanine anhydride. Modification of amino groups in antibodies prepared against two negatively charged haptens ( p-azobenzenearsonate and p-azobenzephosphonate) resulted in a decrease in the number of antibody combining sites, while the average combining constant for these antibodies was unaffected by the modification procedure. The loss of antibody combining sites could be protected against by the presence of hapten during the alanylation reaction. The active sites of antibodies against two other negatively charged haptens ( p-azobenzoate and p-azobenzenesulfonate) were little affected by modification of protein amino groups. The extent of participation of amino groups in the antibody sites seems to vary to a large extent within a population of antibody molecules made against a single hapten. Anti- p-azobenzenearsonate antibody after papain digestion was separated into four Fab fractions with different properties by column chromatography, and the effect of amino group modification on these four fractions varied considerably. The loss in binding activity was not due to conformational changes following modification of amino groups outside of the antibody combining sites since loss of activity could be prevented by the presence of hapten during the alanylation, and since modification of amino groups in antibodies prepared against a neutral hapten (3-azopyridine) and against a positively charged hapten ( p-azophenyltrimethylammonium) resulted in no change in either the average binding constant or the number of antibody combining sites. These results show that amino groups contribute to the positive charge in the combining sites of at least some antibody molecules directed against certain nharged haptens and appear to be absent or not available for alanylation in the active sites of antibody molecules directed against some other negatively charged, positively charged, or neutral haptens.