Sphingosine 1-phosphate (S1P) from mononuclear phagocytes and platelets signals T cells predominantly through S1P1 G protein-coupled receptors (Rs) to enhance survival, stimulate and suppress migration, and inhibit other immunologically relevant responses. Cellular S1P1 Rs and their signaling functions are rapidly down-regulated by S1P, through a protein kinase C (PKC)-independent mechanism, but characteristics of cell-surface re-expression of down-regulated S1P1 Rs have not been elucidated. T cell chemotactic responses (CT) to 10 and 100 nm S1P and inhibition of T cell chemotaxis to chemokines (CI) by 1 and 3 microm S1P were suppressed after 1 h of preincubation with 100 nm S1P, but recovered fully after 12-24 h of exposure to S1P. Late recovery of down-regulated CT and CI, but not early down-regulation, was suppressed by PKC and PKCepsilon-selective inhibitors and was absent in T cells from PKCepsilon-null mice. The same PKCepsilon inhibitors blocked S1P-evoked increases in T cell nuclear levels of c-Fos and phosphorylated c-Jun and JunD after 24 h, but not 1 h. A mixture of c-Fos plus c-Jun antisense oligonucleotides prevented late recovery of down-regulated CT and CI, without affecting S1P induction of down-regulation. Similarly, S1P-elicited threonine phosphorylation of S1P1 Rs was suppressed by a selective inhibitor of PKCepsilon after 24 h, but not 1 h. Biochemical requisites for recovery of down-regulated S1P1 Rs thus differ from those for S1P induction of down-regulation.