Sphingomyelin (SM) biosynthesis is believed to occur in the early Golgi apparatus, plasma membrane and recycling endosomes. In the present study, the localization of the SM synthesis that follows its hydrolysis upon activation of the SM signal-transduction pathway was investigated in human skin fibroblasts treated with tumour necrosis factor (TNF) alpha. After TNFalpha-induced degradation, the intracellular SM levels returned to baseline levels within 30-60 min in cells treated at 37 degrees C. Pretreatment or co-incubation of cells with bacterial sphingomyelinase or phospholipase C, decreasing the SM and phosphatidylcholine content in the external leaflet of the plasma membrane respectively, did not inhibit SM resynthesis. However, SM resynthesis was not observed when TNFalpha-treated cells were continuously exposed to exogenous sphingomyelinase, suggesting that under these particular conditions the resynthesized SM becomes accessible to the enzyme. Furthermore, whereas inhibition of vesicular traffic/endocytosis at 4 degrees C blocked exoplasmic SM resynthesis, it did not alter SM resynthesis in TNFalpha-treated fibroblasts, negating the role of endosomes and the Golgi apparatus. This was further evidenced by the finding that after SM resynthesis, TNFalpha was again able to promote SM turnover, even at 4 degrees C. In addition, when the exoplasmic leaflet SM was hydrolysed by treating fibroblasts with bacterial sphingomyelinase, resynthesis of SM occurred at 37 degrees C much more slowly than after TNFalpha treatment. These findings support strongly the conclusion that the SM, which is resynthesized after TNFalpha-induced hydrolysis, resides in the cytosolic leaflet of the plasma membrane, and that the process involved in this resynthesis displays characteristics different from those of the previously described SM synthases.