1. The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM. This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release. Tension was at 67% of its isometric level by the time 150 microM Pi was released. ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C. The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1. 4. On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl-EGTA. At 5 degrees C the ATPase rate was 5.8 and 4.0 s-1 in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pi on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0.5 mM) decreased the initial ATPase rate by 23%. Pi (10 mM) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5 degrees C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s-1 during the first turnover (based on 180 microM myosin subfragment 1). Tension was 74% of its isometric level by the time 180 microM Pi was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached. Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.