Structurally related sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), may be potential mitogenic factors in the development of vascular disease. The aim of this study was examine the intracellular pathways regulated by S1P, in comparison to SPC, in rat cerebral artery smooth muscle. Male Sprague Dawley rats (6 weeks old, 300-350 g) were humanely killed. Denuded rat cerebral arteries were stimulated with either 5 μM S1P or 10 μM SPC and homogenates subjected to SDS-polyacrylamide electrophoresis followed by immunoblotting. S1P significantly increased extracellular signal-regulated kinase (ERK)1/2 phosphorylation (5.2 ± 1.4-fold increase in band density compared to controls, mean ± s.e.m., n=6, p<0.05) but did not activate p38 mitogen-activated protein kinase (p38MAPK) as assessed with phospho-specific antibodies. In contrast, SPC significantly increased p38MAPK phosphorylation (3.0 ± 0.3-fold increase, n=6, p<0.05) but did not stimulate ERK1/2 activation. This differential activation was confirmed by measuring phosphorylation of heat shock protein (HSP) 27, a known target of p38MAPK. Only SPC, but not S1P, activated HSP27 (2.8 ± 0.3-fold increase, n=5, p<0.05). Dispersed cerebral artery myocytes were prepared by enzymatic digestion and loaded with the fluorescent Ca²⁺ indicator, fura-2. SPC (0.1 – 30 μM) increased [Ca²⁺]_i in a concentration-dependent manner (peak response at 10 μM – 4.1 ± 0.2 ratio 340/380 nm, n=41). In contrast to S1P, the SPC-induced [Ca²⁺]_i increase did not involve release from intracellular stores as determined by a lack of effect of thapsigargin. However, removal of extracellular Ca²⁺ (n=20) or pretreatment with 2 μM nifedipine (n=11) completely blocked the SPC-induced increase in [Ca²⁺]_i (p<0.05). Despite differences in signalling, both S1P and SPC produced increased phosphorylation of the transcription factor cAMP response element-binding protein (CREB) in cerebral artery homogenates as assessed by phospho-specfic antibodies (S1P: 3.2 ± 0.5-fold increase, SPC: 3.3 ± 0.8, n=5). S1P-induced CREB activation was significantly inhibited by pretreatment with PD98059 (ERK1/2 inhibitor) and the Ca²⁺-calmodulin-dependent protein (CaM) kinase inhibitor KN93. CREB activation by SPC was not inhibited by PD98059, but was inhibited by SB203580 (p38MAPK inhibitor) and KN93. In conclusion, S1P and SPC activate distinct members of the MAP kinase family and increase [Ca²⁺]_i via different mechanisms in rat cerebral artery. This does not affect the ability of either S1P or SPC to activate CREB, although this occurs via different pathways.