The rate of relaxation from steady-state force in rabbit psoas fiber bundles was examined before and after phosphorylation of myosin regulatory light chain (RLC). Relaxation was initiated using diazo-2, a photolabile Ca2+ chelator that has low Ca2+ binding affinity (KCa=4.5×105M−1) before photolysis and high affinity (KCa=1.3×107M−1) after photolysis. Before phosphorylating RLC, the half-times for relaxation initiated from 0.27±0.02, 0.51±0.03, and 0.61±0.03 Po were 90±6, 140±6, and 182±9ms, respectively. After phosphorylation of RLC, the half-times for relaxation from 0.36±0.03 Po, 0.59±0.03 Po, and 0.65±0.02 Po were 197±35ms, 184±35ms, and 179±22ms. This slowing of relaxation rates from steady-state forces less than 0.50 Po was also observed when bundles of fibers were bathed with N-ethylmaleimide-modified myosin S-1, a strongly binding cross-bridge derivative of S1. These results suggest that phosphorylation of RLC slows relaxation, most likely by slowing the apparent rate of transition of cross-bridges from strongly bound (force-generating) to weakly bound (non-force-generating) states, and reduces or eliminates Ca2+ and cross-bridge activation-dependent changes in relaxation rates.