CTP:phosphocholine cytidylyltransferase (CCTalpha) is a rate-regulatory enzyme required for phosphatidylcholine (PtdCho) synthesis. CCTalpha is also a phosphoenzyme, but the physiologic role of kinases on enzyme function remains unclear. We report high-level expression of two major isoforms of the c-Jun N-terminal kinase family (JNK1 and JNK2) in murine lung epithelia. Further, JNK1 and JNK2 phosphorylated purified CCTalpha in vitro, and this was associated with a dose-dependent decrease (approximately 40%) in CCT activity. To evaluate JNK in vivo, lung epithelial cells were infected with a replication defective adenoviral vector encoding murine JNK2 (Adv-JNK2) or an empty vector. Adv-JNK2 infection, unlike the empty vector, markedly increased JNK2 expression concomitant with increased incorporation of [32P]orthophosphate into endogenous CCTalpha. Although Adv-JNK2 infection only modestly reduced CCT activity, it reduced PtdCho synthesis by approximately 30% in cells. These observations suggest a role for JNK kinases as negative regulators of phospholipid synthesis in murine lung epithelia.