Adenosine 5'-triphosphate (ATP) derivatives of the types N6-R-ATP [R = (CH2)nHNCOCH2I, (CH2)nNHCO-(CH2)mHNCOCH2I, or (CH2)nCON(Me)(CH2)mN(Me)CO(CH2)nNHCOCH2I], N6-Me-N6-R-ATP [R = (CH2)nN-(Me)CO(CH2)mNHCOCH2I], and 8-R-ATP [R = NH(CH2)nNHCOCH2I] with 5--19 spacer atoms between N6 or C-8 and iodine have been evaluated as substrates, reversible inhibitors, and inactivators of adenylate kinase (AK). With Escherichia coli AK, the derivatives were noncompetitive inhibitors, Ki = 4.7--7.3 mM, with little affinity for the ATP site, and N6-(CH2)nNHCOCH2[-ATP (n = 5 or 6) effected progressive inhibitions that were not ATP site directed. With rat muscle AK (M-AK), some compounds had slight affinity for the ATP site as evidenced by weak substrate activity with as much as 8 spacer atoms, but all compounds tested were weak noncompetitive inhibitors; Ki = 6--12 mM vs. ATP. The ATP derivatives, notably N6-(CH2)8NHCOCH2I-ATP, mediated a progressive inhibition of M-AK, which was abolished by substitution of hydrogen for the iodine and thus presumably involves alkylation of the enzyme. The inhibition appeared not to be ATP site directed because kinetic analysis indicated a random bimolecular enzyme-inhibitor reaction and because N6-(CH2)8NHCOCH2I-AMP and its adenosine counterpart, which have relatively low affinity for the ATP site, were more effective than N6-(CH2)8NHCOCH2I-ATP. The ATP derivatives were substrates (KM = 0.4--1.6 mM) and/or competitive inhibitors (Ki = 0.3--6.2 mM) vs. ATP of rat isozymes AK II or III. Exposure of AK II or III for 6 h, 22 degrees C, at pH 7.6 to 10 mM levels of the 1:1 Mg complexes of 25 of the ATP derivatives led in no case to progressive enzyme inhibition, suggesting the absence near the ATP sites of nucleophilic groups suitably positioned for alkylation.