A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr⁷⁰⁹, and two other sites at Thr⁶⁹⁵ and Thr⁴⁹⁵. One of the sites for cAMP-dependent protein kinase (PKA) was Ser⁶⁹⁴. Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr⁶⁹⁵→Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr⁶⁹⁵ was the major inhibitory site, Thr⁷⁰⁹ had only a slight inhibitory effect and Ser⁶⁹⁴ had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.