Abstract The influence of sodium dodecyl sulfate (SDS) on ADP-ribosylation by Clostridium botulinum C3 exoenzyme (C3) was studied. SDS increased the ADP-ribosylation of recombinant rhoA and human platelet cytosolic proteins maximally at 0.01% whereas higher concentrations of the detergent (>0.01%) inhibited the ADP-ribosylation. In contrast, ADP-ribosylation of human platelet membranes and of recombinant rhoB was inhibited by the detergent. The K m for NAD of the ADP-ribosylation of rhoA was decreased by SDS from about 10 to 0.6 μM. Whereas in the absence of SDS, the C3-induced ADP-ribosylation of recombinant rhoA is not affected by the amphiphilic wasp venom mastoparan, in the presence of SDS (0.01%) mastoparan (100 μM) inhibited the ADP-ribosylation. C3-associated NAD-glycohydrolase activity was maximally and half-maximally inhibited by 0.1 and 0.013% SDS, respectively. Inhibition of NAD-glycohydrolase activity was reversed by diluting out SDS indicating that C3 was not irreversibly denatured by SDS treatment. SDS (0.01%) completely inhibited the [ 3H]GTP binding of rhoA whereas the release of previously bound nucleotide was not affected. The data indicate that changes in the lipophilicity of rhoA protein largely affect its ability to serve as a substrate for C3-like ADP-ribosyltransferases.