We have shown previously that N-[2-bromocinnamyl(amino)-ethyl]-5-isoquinolinesulphonamide (H-89), a selective inhibitor of cyclic-AMP-dependent protein kinase (PKA), inhibits phosphatidylcholine biosynthesis in HeLa cells. In the present study, we elucidated the mechanism underlying the described inhibition. Treatment of cells with 10 microM H-89 had no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase. However, H-89 slightly affected the distribution of cytidylyltransferase between cytosol and membranes, but the cellular 1,2-diacylglycerol content was not influenced. Furthermore, pulse-chase experiments revealed that H-89 did not affect cytidylyltransferase activity. Instead, H-89 inhibited choline kinase, the enzyme catalysing the first step in the CDP-choline pathway. In the presence of 10 microM H-89, choline kinase activity was inhibited by 36 +/- 7.6% in vitro. Additionally, the phosphorylation of choline to phosphocholine was inhibited by 30 +/- 3% in cell-culture experiments. This inhibitory effect could be partly prevented by simultaneous addition of 10 microM forskolin, indicating that choline kinase is regulated in part by PKA activity.