In skeletal muscle, there is a length dependence of staircase potentiation for which the mechanism is unclear. In this study we tested the hypothesis that abolition of this length dependence by caffeine is effected by a mechanism independent of enhanced Ca2+release. To test this hypothesis we have used caffeine, which abolishes length dependence of potentiation, and dantrolene sodium, which inhibits Ca2+release. In situ isometric twitch contractions of rat gastrocnemius muscle before and after 20 s of repetitive stimulation at 5 Hz were analyzed at optimal length (Lo), Lo- 10%, and Lo+ 10%. Potentiation was observed to be length dependent, with an increase in developed tension (DT) of 78 ± 12, 51 ± 5, and 34 ± 9% (mean ± SEM), at Lo- 10%, Lo, and Lo+ 10%, respectively. Caffeine diminished the length dependence of activation and suppressed the length dependence of staircase potentiation, giving increases in DT of 65±13, 53 ± 11, and 45 ± 12% for Lo- 10%, Lo, and Lo+ 10%, respectively. Dantrolene administered after caffeine did not reverse this effect. Dantrolene alone depressed the potentiation response, but did not affect the length dependence of staircase potentiation, with increases in DT of 58 ± 17, 26 ± 8, and 18 ± 7%, respectively. This study confirms that there is a length dependence of staircase potentiation in mammalian skeletal muscle which is suppressed by caffeine. Since dantrolene did not alter this suppression of the length dependence of potentiation by caffeine, it is apparently not directly modulated by Ca2+availability in the myoplasm.