Protein phosphatase 1 (PP1) catalytic subunits dephosphorylate specific substrates in discrete subcellular compartments to modulate many cellular processes. Canonical PP1-binding motifs (R/K-V/ I-X-F) in a family of proteins mediate subcellular targeting, and the amino acids that form the binding pocket for the canonical motif are identical in all PP1 isoforms. However, PP11 but not PP1 is selectively localized to F-actin-rich dendritic spines in neurons. Although the F-actin-binding proteins neurabin I and spinophilin (neurabin II) also bind PP1, their role in PP1 isoform selective targeting in intact cells is poorly understood. We show here that spinophilin selectively targets PP11, but not PP1, to F-actin-rich cortical regions of intact cells. Mutation of a PP11 selectivity determinant (N⁴⁶⁴EDYDRR ⁴⁷⁰ in spinophilin: conserved as residues 473–479 in neurabin) to VKDYDTW severely attenuated PP11 interactions with neurabins in vitro and in cells and disrupted PP11 targeting to F-actin. This domain is not involved in the weaker interactions of neurabins with PP1. In contrast, mutation of the canonical PP1-binding motif attenuated interactions of neurabins with both isoforms. Thus, selective targeting of PP11 to F-actin by neurabins in intact cells requires both the canonical PP1-binding motif and an auxiliary PP11-selectivity determinant.—Carmody, L. C., Baucum II, A. J., Bass, M. A., Colbran, R. J. Selective targeting of the 1 isoform of protein phosphatase 1 to F-actin in intact cells requires multiple domains in spinophilin and neurabin.