Increasing the intramuscular stores of total creatine [TCr = creatine (Cr) + creatine phosphate (CrP)] can result in improved muscle performance during certain types of exercise in humans. Initial uptake of Cr is accompanied by an increase in cellular water to maintain osmotic balance, resulting in a decrease in myoplasmic ionic strength. Mechanically skinned single fibers from rat soleus (SOL) and extensor digitorum longus (EDL) muscles were used to examine the direct effects on the contractile apparatus of increasing [Cr], increasing [Cr] plus decreasing ionic strength, and increasing [Cr] and [CrP] with no change in ionic strength. Increasing [Cr] from 19 to 32 mM, accompanied by appropriate increases in water to maintain osmolality, had appreciable beneficial effects on contractile apparatus performance. Compared with control conditions, both SOL and EDL fibers showed increases in Ca(2+) sensitivity (+0.061 +/- 0.004 and +0.049 +/- 0.009 pCa units, respectively) and maximum Ca(2+)-activated force (to 104 +/- 1 and 105 +/- 1%, respectively). In contrast, increasing [Cr] alone had a small inhibitory effect. When both [Cr] and [CrP] were increased, there was virtually no change in Ca(2+) sensitivity of the contractile apparatus, and maximum Ca(2+)-activated force was approximately 106 +/- 1% compared with control conditions in both SOL and EDL fibers. These results suggest that the initial improvement in performance observed with Cr supplementation is likely due in large part to direct effects of the accompanying decrease in myoplasmic ionic strength on the properties of the contractile apparatus.