We have measured the steady state kinetics of hydrolysis and presteady state kinetics of binding of the nucleoside triphosphate GTP, CTP, aza-ATP (1-N6-etheno-2-aza-ATP), and ATP by rabbit skeletal actomyosin-S1. The maximum rates of steady state hydrolysis at 10 degrees C at low ionic strength are: CTP, 1.9 s-1 > ATP, 1.3 s-1 > aza-ATP, 0.19 s-1 > GTP, 0.03 s-1. A similar dependence of the rate of steady state hydrolysis upon nucleotide structure has been observed in isometrically contracting muscle fibers in the accompanying paper (Pate, E., Franks-Skiba, K., White, H., and Cooke, R. (1993) J. Biol. Chem. 268, 10046-10053) which strongly suggests that the same biochemical step that limits the maximum rate of hydrolysis of nucleoside triphosphates by actomyosin-S1 in solution also limits the rate of hydrolysis by isometrically contracting muscle fibers. The apparent second order rate constants for the dissociation of actomyosin-S1 by nucleoside triphosphates at 10 degrees C are: ATP, 2.7 x 10(6) M-1 s-1 > aza-ATP, 3.4 x 10(5) M-1 s-1 > GTP, 2.5 x 10(5) M-1 s-1 > CTP, 1.4 x 10(5) M-1 s-1. There is an excellent correlation between the second order rate constant for the dissociation of actomyosin-S1 in solution and the dependence of shortening velocity in glycerinated muscle fibers upon the concentration for ATP, aza-ATP, and CTP (as per accompanying article; Pate et al., 1993). We have used the second order rate constants obtained in solution for the dissociation of actomyosin-S1 by these nucleotides and shortening velocity data obtained with the same nucleoside triphosphates in glycerinated psoas fibers in the accompanying article (Pate et al., 1993) to determine the average distance over which cross-bridges remain attached during unloaded shortening to be 5-12 nm.