The synthesis and metabolic fate of purine nucleotides were studied, employing labeled precursors, in primary rat muscle cultures. The cultures were found to produce purine nucleotides, by de novo and salvage pathways, both exhibiting dependence on cellular availability of substrate 5-phosphoribosyl-1-pyrophosphate (PPRibP). Depletion of cellular PPRibP decelerated the rate of purine synthesis, whereas increasing PPRibP generation by high Pi concentration in the incubation medium, accelerated purine synthesis. Ribose accelerated purine synthesis, indicating that ribose 5-phosphate availability in the cultured muscle is limiting for PPRibP synthesis. The study in the muscle cultures of the metabolic fate if IMP formed from [14C]formate and that of nucleotides formed from labeled purine bases, revealed that the main flow in the nucleotide interconversions pathways is from AMP to IMP. The flow from IMP to GMP and to AMP appeared to be of a lesser magnitude and virtually no flow could be detected from GMP to IMP. The greatest proportion of radioactivity of purine nucleotides following synthesis by either de novo or salvage pathways, accumulated in IMP, reflecting the relative rates of flows between the various nucleotides and probably also a relatively low, or inhibited activity of the IMP nucleotidase. The results suggest that primary muscle cultures are a plausible model for the study of the role of purine metabolism in muscle work.