Two isoforms of sphingosine kinase (SphK) catalyze the formation of sphingosine-1-phosphate (SIP). Whereas, SphKl stimulates cell growth and survival, it was found that when overexpressed in mouse NIH 3T3 fibroblasts SphK2 enhances caspase-dependent apoptosis in response to serum deprivation, independently of S1P receptors. Sequence analysis revealed that SphK2 contains a 9 amino acid motif similar to that present in BH3-only proteins. Studies showed that the BH3-only domain, catalytic activity, endoplasmic reticulum (ER) stress, and uptake of calcium by the mitochondria may all contribute to the apoptotic effects of overexpressed SphK2 in NIH 3T3 cells. Further studies in human carcinoma cells showed that overexpression of SphK2 increased the expression of the cyclin dependent kinase (cdk) inhibitor p21, but interestingly had no effect on p53 or its phosphorylation. Correspondingly, downregulation of endogenous SphK2 with small interfering RNA (siRNA) targeted to unique mRNA sequences decreased basal and doxorubicin-induced expression of p21 without affecting p53. In addition, downregulation of SphK2 decreased G2/M arrest in response to doxorubicin. Surprisingly however, siSphK2 markedly enhanced apoptosis induced by doxorubicin in MCF7 and HCT-116 cells. This result raises the question of how overexpression of SphK2 decreases cell growth and enhances apoptosis while its downregulation sensitizes cells to apoptosis. A partial answer may come from the possibility that when SphK2 is overexpressed it does not always have the same subcellular distribution as the endogenous protein. It may also be possible that proteolysis of overexpressed SphK2 might induce apoptosis due to liberation of its BH3 peptide domain, which does not occur at the levels at which endogenous SphK2 is expressed. Collectively, these results demonstrate that endogenous SphK2 is important for p53-independent induction of p21 expression by doxorubicin and suggest that SphK2 expression may influence the balance between cytostasis and apoptosis.