We have previously provided evidence suggesting that phosphatidic acid, possibly derived from the hydrolysis of phosphatidylcholine by phospholipase D (PLD), is involved in platelet-derived growth factor (PDGF)-mediated increases in extracellular signal-regulated kinase (ERK) activity and DNA synthesis in rat hepatic stellate cells (HSC), the primary fibrogenic cells of the liver. A recent study has shown the presence of P2Y nucleotide receptors on HSC that are coupled to contraction and synthesis of the matrix component, alpha(1)-procollagen, leading to the suggestion that they may represent a new therapeutic target in the treatment of liver fibrosis. However, although extracellular nucleotides have been shown to stimulate both PLD and ERK, and to elicit proliferation of fibrogenic cells outside the liver, their effect on these parameters in HSC have not yet been investigated. PLD activity was determined by [(3)H]choline release and [(3)H]phosphatidylbutanol production, ERK activity by Western blotting, and DNA synthesis by [(3)H]thymidine incorporation. We report here, for the first time in HSC, that extracellular nucleotides stimulate PLD activity and a sustained activation of ERK. However, in contrast to PDGF, nucleotides had negligible effects on DNA synthesis. Moreover, the effects of PDGF and nucleotides on PLD and ERK were not additive, suggesting activation of the same PLD isoform and pool of ERK. The data demonstrate that nucleotide-stimulated PLD and ERK activities are not coupled to DNA synthesis in HSC. Instead, these responses may be linked to other phenotypic changes associated with activated HSC such as increases in contraction, motility, or extracellular matrix deposition.