We have previously shown that acute exercise increases the level of sphingosine-1-phosphate (S1P) in plasma and ceramide in erythrocytes of untrained subjects. The aim of the current study was to examine the effect of ultramarathon run on the plasma and erythrocyte levels of the following bioactive sphingolipids: S1P, sphinganine-1-phosphate (SA1P), sphingosine, sphinganine, and ceramide. Blood samples were collected from seven male amateur runners participating in a 48-hr ultramarathon race before the run, after 24 and 48 hr of running, and following 24 and 48 hr of recovery. The sphingolipids were quantified by means of HPLC. Sustained running for 48 hr resulted in a progressive decline in plasma S1P to a level significantly lower than at prerace, and then remained stable over the next 48 hr of recovery. In erythrocytes, S1P content was stable until 24 hr of recovery, then rose abruptly to reach peak values after 48 hr of recovery. The plasma level of SA1P decreased progressively during the competition and remained unchanged over the recovery. In erythrocytes, the level of SA1P increased after 24 hr running and normalized thereafter. The level of ceramide, both in plasma and erythrocytes, was not significantly affected by the ultraendurance run. We speculate that reduction in plasma level of S1P during and after the run reduces its biological actions and might be responsible for some negative side-effects of the ultraendurance effort.