Recent evidence suggests that sphingosine 1-phosphate (S1P) regulates self-renewal of human embryonic stem (ES) cells and differentiation of mouse embryoid bodies (derived from mouse ES cells) to cardiomyocytes. We have investigated the role of S1P in regulating ERK-1/2 signaling in mouse ES cells. In this regard, we found that both mouse ES-D3 and CGR8 cells express S1P(1), S1P(2), S1P(3), and S1P(5) but lack S1P(4). The treatment of ES cells with S1P induced the activation of ERK-1/2 via a mechanism that was not mediated by S1P(1), S1P(2), or S1P(3). This was based on: (i) the failure of S1P(1), S1P(2), or S1P(3) antagonists to inhibit S1P-stimulated ERK-1/2 activation and (ii) the failure of SEW 2871 (S1P(1) receptor agonist) to stimulate ERK-1/2 activation. The treatment of ES cells with phytosphingosine 1-phosphate (phyto-S1P), which we show here is an agonist of the S1P(5) receptor, stimulated ERK-1/2 activation. These findings therefore suggest that S1P(5) may mediate the effects of S1P in terms of regulating ERK-1/2 signaling in ES cells. The S1P-dependent activation of ERK-1/2 was sensitive to inhibition by pertussis toxin (uncouples the G-protein, G(i) from GPCR), bisindolylmaleimide I (PKC inhibitor), and PP2 (c-Src inhibitor), but was not reduced by LY29004 (PI3K inhibitor) suggesting that S1P uses G(i)-, PKC-, and c-Src-dependent mechanisms to activate the ERK-1/2 pathway in ES cells.