It is known that a range of firing frequencies can be observed during in vivo muscle activity, yet information is lacking as to how different in vivo-like frequencies may affect force generation of skeletal muscle. This study examined the effects of constant (CSF, constant within one contraction) and decreasing stimulation frequencies (DSF) on mean sarcomere length-force characteristics of rat gastrocnemius medialis fibre bundles. The CSF resulted in an optimal mean sarcomere length (lso) of 2.30 (SEM 0.02), 2.46 (SEM 0.03), 2.76 (SEM 0.03) and more than 2.99 (SEM 0.07) lm, for 100, 50, 30 and 15 Hz, respectively. Compared to 100-Hz stimulation, both lso and the ascending limb of the relationship significantly shifted to higher lengths with lower frequencies. No shift was encountered for the initial part of the descending limb. The DSF reduced the frequency-induced shift to higher mean lengths [lso 2.33 (SEM 0.02), 2.52 (SEM 0.08) and more than 2.92 (SEM 0.10) microm, respectively, for 50, 30 and 15 Hz]. No effect of activation time on length-force characteristics was observed. It was concluded from these studies that the frequency and history of stimulation is a major determinant of the length-force characteristics of muscle fibre bundles, and should be taken into account when analysing animal and human locomotion. The previously observed frequency-induced shift in whole muscle length-force relationship resides mainly at the level of fibre bundles.