Elevators of cAMP, such as prostaglandin E2 (PGE2), activate protein kinase A (PKA) and induce PKA-stimulated motility and metastasis by metastatic Lewis lung carcinoma cells (LLC-LN7). Non-metastatic LLC (LLC-C8) are unresponsive to cAMP elevation even though they are not deficient in the PKA enzymes. To determine whether this PKA unresponsiveness might be due to increased dephosphorylation by serine/threonine protein phosphatases (PP-1/2A) within non-metastatic LLC-C8, the effects of the PP-1/2A inhibitor okadaic acid on the migration and invasion by non-metastatic LLC-C8 cells was measured. Okadaic acid stimulated motility of non-metastatic LLC-C8 cells to a level that was comparable to that of metastatic LLC-LN7 cells. PGE2 further increased the motility of the non-metastatic LLC-C8 cells when okadaic acid was present, although not in the absence of okadaic acid. The stimulation of motility by okadaic acid was diminished when PKA activity was inhibited. Dose-response studies with concentrations of okadaic acid that selectively inhibited PP-2A or both PP-2A and PP-1 showed a progressive increase in migration of non-metastatic LLC-C8 cells, suggesting that both PP-1 and PP-2A limit their motility. By contrast, metastatic LLC-LN7 cells were more motile than were non-metastatic LLC-C8 cells, but this motility was only marginally affected by okadaic acid. Comparisons of the levels of PP-1/2A enzyme activities in the LLC variants showed more activity in non-metastatic LLC-C8 than in metastatic LLC-LN7 cells. The identity of the PP whose activity was increased in the non-metastatic LLC-C8 was assessed by using okadaic acid, which selectively inhibits PP-2A activity at low concentrations and PP-1 and PP-2A at high concentrations, and calyculin A, which inhibits PP-2A at a similar concentration to that affected by okadaic acid but is more potent at inhibiting PP-1. The inhibition of PP activities by okadaic acid and by calyculin A showed a pattern which suggested the presence both of PP-1 and of PP-2A in non-metastatic LLC-C8 cells, but the presence of PP-1 and a reduction in PP-2A in metastatic LLC-LN7 cells. The sum of these data suggests that PKA-stimulated motility is restricted both by PP-1 and by PP-2A in non-metastatic LLC, and that a deficiency in this restriction results in increased migration and invasion.