To determine whether thiophosphorylation of the 20-kDa myosin light chain activates each head of smooth muscle myosin independently of the head with which it is paired, chicken gizzard smooth muscle myosin was randomly thiophosphorylated, producing a mixture of unphosphorylated and singly and doubly thiophosphorylated myosin. Thiophosphorylation levels were measured by glycerol-urea gels, and the activity of this myosin was determined by actin-activated adenosinetriphosphatase measurements and in an in vitro motility assay, where the velocity of actin filaments moving over a myosin-coated surface is measured. Activity at each thiophosphorylation level was similar to that previously observed for mixtures of unphosphorylated and doubly thiophosphorylated myosin (D. E. Harris, S. S. Work, R. K. Wright, N. R. Alpert, and D. M. Warshaw. J. Muscle Res. Cell Motil. 15: 11-19, 1994). All doubly thiophosphorylated myosin was then formed into filaments and removed from randomly thiophosphorylated myosin by centrifugation. The remaining myosin (mixture of unphosphorylated and singly phosphorylated myosin), which could not polymerize because of their conformation, retained approximately 70% activity compared with mixtures of unphosphorylated and doubly thiophosphorylated myosin. Thus a thiophosphorylated smooth muscle myosin head can produce substantial biochemical and mechanical activity, even when it is paired with an unphosphorylated partner.