The substrate specificities of N-acetyltransferases (NAT) were investigated by measuring Vmax and Km values with p-aminobenzoic acid (I), p-aminobenzamide (II), p-amino-benzamidopyridine (III), 2-(p-aminobenzamido)-4,6-dimethylpyrimidine (VI), and the corresponding p-aminobenzenesulfonamides (VII, VIII and XI) using at rat liver and blood preparations. With liver NAT, II, III and VI had lower Km and higher Vmax values than did their corresponding sulfonyl analogs (VII, VIII, XI). III was extraordinarily active (Vmax 854 nmol/mg protein/h); in contrast, II gave a Vmax of 22.4. Sulfadiazine (IX) and sulfamerazine (X) were acetylated at a very slow rate. The activities of the blood enzymes on these compounds were very different. The Vmax values obtained with blood NAT for II, VI and VII were sharply decreased. Surprisingly, acetylation of III, VIII, IX and X could not be detected. In contrast to liver, the blood NAT gave lower values of both Vmax and Km for the S analog of II and a much higher Km for I. While p-aminobenzoic acid was the best substrate for blood NAT, substitution of the amido nitrogen of p-aminobenzamide with an aromatic substituent enhanced the substrate potential for liver NAT, III may be useful as a substrate for the rapid classification of slow and fast acetylators.