The ATP analogues 6-(n-butylamino)-, 6-(di-n-butylamino)-, and 6-(n-butylthio)-9-β-D-ribofuranosylpurine 5' triphosphate have been synthesized and studied as inhibitors and/or substrates of the rat muscle adenylate kinase isozyme (AK M) and the rat liver isozymes AK II and III. The 6-NH(n-Bu) and 6-S(n-Bu) analogues were substrates (Vmax relative to ATP, 13-190%) of the three AK isozymes, whereas the 6-N(n-Bu)2 analogue was a weak substrate and a competitive inhibitor of AK M and AK III. The affinities of the analogues relative to ATP [KM (ATP)/KM or Ki] were 0.03-0.075 for AK III and 0.14-0.28 for AK M, and affinities for AK M exceeded those for AK III by factors of 2.3-7.0. P1,P5-Di(adenosine-5') pentaphosphate (Ap5A) was synthesized by an improved method and was found to be a potent two-site inhibitor (Ki = 0.28 μM), competitive toward AMP or ATP, for the three AK isozymes. 8-SEt-Ap5A also behaved as a two-site inhibitor; the 8-SEt group reduced the affinity for AK M 12-fold but increased the affinity for AK II and III 4-fold, resulting in ca. 45-fold more effective inhibition of AK II and III (Ki = 0.07 μM) than of AK M (Ki = 3.25 μM). The 8-SEt group of 8-SEt-ATP likewise reduced affinity for the ATP site of AK M but enhanced affinity for the ATP sites of AK II and III, resulting in at least 30-fold more effective inhibition of AK II and III. 8-SEt-AMP inhibited AK II and III noncompetitively (Ki = 21-24 mM) with respect to AMP, indicating that the 8-(ethylthio)adenosine moiety of 8-SEt-Ap5A probably binds to the ATP sites of these isozymes. 8-SEt-Ap5A had ca. 1000-fold more affinity for AK II or III than did 8-SEt-ATP. The findings indicate that isozyme-selective inhibitory effects of a substrate derivative can be imparted to a two-site inhibitor, leading to significant enhancement of inhibitory potency.