Derivatives of adenosine 5'-diphosphate (ADP) with a substituent of 1-4 atoms at any of eight positions have been synthesized and evaluated as substrates and inhibitors of the liver (L), muscle (M), and kidney (K) isozymes of rat pyruvate kinase (PK). Inhibitory potencies of the compounds were expressed as KM (ADP)/Ki or as KM (ADP)/KM when no Ki value was available. Nine of 14 ADP derivatives exhibited differential inhibitions. The M and K isozymes, which cross-react immunologically with each other but not with the L form, were inhibited differentially by 5 of the 14 derivatives. PK-K was preferentially inhibited by two derivatives, PK-L by three derivatives, and PK-M by two derivatives. Among the most selective and/or effective inhibitors were 3'-OMe-ADP [KM (ADP)/Ki = 0.07 with PK-K; inhibitory potency, K/M/L, 7.6:6.0:1], N6-Me,N6-(CH2)4N(Me)COMe-ADP (prepared previously) [KM (ADP)/KM = 0.43 with PK-L; inhibitory potency, L/K/M, 3:2:1], and 8-NHEt-ADP [KM (ADP)/Ki = 1.0 with PK-M; inhibitory potency, M/K/L, 7.1:1.2:1]. These and previous studies with two other enzymes indicate that monosubstituted substrate derivatives that bear short substituents (usually 1-4 atoms) at various positions are potentially useful probes in early stages of the attempted design of isozyme-selective inhibitors.