We have used a scallop hybrid myosin test system in an attempt to determine the regulatory properties of an individual myosin II isoform from rat brain. The complete coding region of cDNA corresponding to a regulatory light chain isoform previously shown to be expressed in brain [Feinstein, Durand and Milner (1991) Mol. Brain Res. 10, 97-105] was ligated within the prokaryotic expression vector, pAED4, overexpressed in bacteria, and the purified light chain incorporated within a scallop hybrid myosin. Actin activation was calcium insensitive for all hybrids tested, irrespective of whether light chain phosphorylation had taken place before, or subsequent to, hybrid formation. We discuss the implications of these results, including the possibility that these results constitute evidence for a myosin II isoform within brain that is regulated at the level of the thin filament. In addition, evidence is presented for the presence of an additional, novel isoform of regulatory light chain expressed in rat brain.