The specificity of the catalytic subunit of protein phosphatase-1 (PP1c) is modified by regulatory subunits that target it to particular subcellular locations. Here, we identify PP1c-binding domains on GL and GM, the subunits that target PP1c to hepatic and muscle glycogen, respectively, and on M110, the subunit that targets PP1c to smooth muscle myosin. GM-(G63-T93) interacted with PP1c and prevented GL from suppressing the dephosphorylation of glycogen phosphorylase, but it did not dissociate GL from PP1c or affect other characteristic properties of the PP1GL complex. These results indicate that GL contains two PP1c-binding sites, the region which suppresses the dephosphorylation of glycogen phosphorylase being distinct from that which enhances the dephosphorylation of glycogen synthase. At higher concentrations, GM-(G63-N75) had the same effect as GM-(G63-T93), but not if Ser67 was phosphorylated by cyclic-AMP-dependent protein kinase. Thus, phosphorylation of Ser67 dissociates GM from PP1c because phosphate is inserted into the PP1c-binding domain of GM. M110-(M1-E309) and M110-(M1-F38), but not M110-(D39-E309), mimicked the M110 subunit in stimulating dephosphorylation of the smooth muscle myosin P-light chain and heavy meromyosin in vitro. However, in contrast to the M110 subunit and M110-(M1-E309), neither M110-(M1-F38) nor M110-(D39-E309) suppressed the PP1c-catalysed dephosphorylation of glycogen phosphorylase. These observations suggest that the region which stimulates the dephosphorylation of myosin is situated within the N-terminal 38 residues of the M110 subunit, while the region which suppresses the dephosphorylation of glycogen phosphorylase requires the presence of at least part of the region 39-309 which contains seven ankyrin repeats. M110-(M1-F38) displaced GL from PP1c, while GM-(G63-T93) displaced M110 from PP1c in vitro. These observations indicate that the region(s) of PP1c that interact with GM/GL and M110 overlap, explaining why different forms of PP1c contain just a single targetting subunit.