To understand the domain requirements of phosphorylation-dependent regulation, we prepared three recombinant constructs of nonmuscle heavy meromyosin IIB containing 1) two complete heads, 2) one complete head and one head lacking the motor domain, and 3) one complete head and one head lacking both motor and regulatory domains. Steady-state ATPase measurements showed that phosphorylation did not alter the affinity for actin by more than a factor of 2 for any construct. Phosphorylation increased V(max) by a factor of 10 for construct 1 and 1.5-3 for construct 2 but had no effect for construct 3. Single turnover measurements, a better measure of slow rates inherent to unphosphorylated regulated myosins, showed that the single-headed construct 2, like construct 3 retains less than 1% of the regulatory properties of the double-headed construct 1 (300-fold activation). Therefore, a complete head cannot be down-regulated by a regulatory domain (without the motor domain) on the partner head. Two motor domains are required for regulation. This result is predicted by a structural model (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390) showing interaction between the motor domains for unphosphorylated smooth muscle myosin, if motor-motor interaction is the basis for down-regulation.