A number of adenosine analogs modified in the imidazole portion of the purine ring or in the carbohydrate moiety have been examined for their ability to inhibit the aggregation of human blood platelets induced by 10 μM ADP. Also, the effects of incorporation of adenosine analogs into the nucleotide pools, or of alteration of the natural adenine nucleotide levels, were studied. In accord with earlier findings, alterations in the ribose moiety of adenosine markedly diminished effectiveness in blocking ADP-induced aggregation. The C-nucleoside, formycin and its 1-methyl and 2-methyl derivatives, displayed little inhibitory activity and α-D-ribofuranosyl adenine and α- l-lyxofuranosyl adenine were without activity. Replacement of the 5'-hydroxyl group of the ribose by carboxyl, amino or S-methyl groups decreased capacity to inhibit aggregation. In contrast, carbocyclic adenosine (wherein an oxygen atom of the ribofuranosyl ring is replaced by a methylene group) retained full ability to inhibit ADP-induced aggregation. The effects of modifications in the imidazole portion of the purine ring of adenosine are complex. Tubercidin (7-deazaadenosine), 4-aminopyrazolo [3,4- d]pyrimidine ribonucleoside (4-APP-ribonucleoside) and 8-azaadenosine displayed negligible or weak inhibitory activity. However, analogs related to tubercidin, i.e. 6-aminotoyocamycin and sangivamycin (5-carboxamide tubercidin), as well as the 3-carboxamide derivative of 4-APP ribonucleoside, displayed inhibitory activity approaching that of adenosine. No relation was established between the incorporation of adenosine analogs into platelet nucleotide pools and their ability to inhibit ADP-induced aggregation. Analogs such as formycin, which in the presence of the adenosine deaminase inhibitor, deoxycoformycin, readily enter the platelet nucleotide metabolic pool with replacement of a substantial portion of the natural adenine nucleotides, cause only weak inhibitory effects, whereas analogs such as 6-aminotoyocamycin, and sangivamycin, which did not form analog nucleotides or formed only small quantities of the 5'-monphosphate nucleotides, display inhibitory activity comparable to that of adenosine. When platelets were incubated with 2-fluoroadenosine, a potent inhibitor of aggregation, large amounts of 2-fluoroadenosine, 5'-mono, di- and triphosphate nucleotides were formed with a coincident marked decrease in the ATP concentrations. When these platelets were washed free of extracellular 2-fluoroadenosine and resuspended in platelet-free plasma, normal aggregation was produced by ADP. However, incubation of this suspension for 10 min with 2-fluoroadenosine resulted in an almost complete inhibition of ADP-induced aggregation.