The mechanism underlying the formation of easily releasable myofilaments, from myofibrils treated with an ATP-containing relaxing solution, was examined in this investigation. The proportion of releasable myofilaments purified from myofibrils of cardiac, fast- and slow-twitch muscles increased as the [ATP] was raised from 0 to 8.5 mM. The protein composition of the easily releasable myofilaments did not differ with increasing ATP concentrations as observed by 5-15% linear gradient SDS-PAGE. There is a nucleotide specificity to the release of myofilaments in the order of ATP greater than GTP much greater than UTP greater than CTP. Experiments with AMP-PNP and inorganic phosphate (Pi) showed that ATP hydrolysis and the build up of Pi are not requirements in the formation of the easily releasable myofilaments. The release of myofilaments was found to be insensitive to variations in pH from 6.5 to 7.5. The ATP stimulation of myofilaments release is ubiquitin-independent, since incubation of purified myofibrils with ubiquitin (1-100 micrograms/ml) at both 20 and 37 degrees C did not change the amount released. Modifying the free sulfhydryl group content by treatment of myofibrils with NEM (0.01-1 mM) or silver nitrate (0.1-10 mM) decreased the proportion of myofilaments that were releasable. Exclusion of 1 mM DTT from the preparation of myofibrils had similar results. These results indicate that the formation of easily releasable myofilaments can be mediated by metabolically related parameters such as the adenosine nucleotides and the reduction-oxidation status of the myofibrillar proteins of striated muscle.