1. After the degradation of cell-surface sphingomyelin (SM) by exogenous sphingomyelinase (SMase), the resynthesis of SM by baby-hamster kidney (BHK) and human leukaemia-60 (HL-60) cells was examined in relation to utilization of substrate phosphatidylcholine (PtdCho) and generation of the expected product, diradylglycerol (DRG). Using [3H]choline-labelled BHK cells incubated in non-radioactive medium, SMase caused a release of phosphocholine, which was derived approximately equally from SM and PtdCho, consistent with the anticipated resynthesis of SM at the expense of PtdCho. However, with choline-labelled cells incubated in radioactive medium or [14C]acetate-labelled cells treated with SMase, no loss of radioactivity from PtdCho or accumulation of labelled DRG was observed, suggesting that any DRG produced as a consequence of SM synthesis must have been rapidly converted back into PtdCho. In contrast, SMase treatment of HL-60 cells caused more than a doubling of DRG levels at the expense of PtdCho, and this appears to be the first demonstration of a rise in DRG related to the synthesis of SM. The DRG produced consisted of about 80% 1,2-diacylglycerol and 18% 1-O-alkyl-2-acylglycerol species, a similar composition to that of the DRG backbone of total cell PtdCho. 2. The requirement for cell-surface PtdCho in the biosynthesis of SM by BHK cells was also investigated. Treatment of [3H]choline-labelled BHK cells with Bacillus cereus PtdCho-specific phospholipase C (PLC) rapidly degraded about 6% of the total PtdCho, which was assumed to represent the cell-surface pool. This did not appear to be the pool of PtdCho required for SM synthesis, since (a) the released phosphocholine was additional to that derived from PtdCho in cells treated with SMase and (b) treatment with PLC did not affect SM synthesis, either de novo or in response to degradation of cell-surface SM by SMase. These findings suggest either that there is no SM synthase in the plasma membrane or, if it is present, then it does not utilize cell-surface PtdCho as a substrate.