The mass levels of bioactive lipids known to modulate signal transduction or to possess other biological activities were measured in HIV-infected CEM cells. The levels of diacylglycerol, an activator of protein kinase C, as well as of alkylacylglycerol were elevated. A more drastic increase was observed in the ceramide levels after HIV-infection, whereas sphingosine levels were hardly influenced. Interestingly, the magnitude of the changes was related to the infection time, being higher at 8 days after infection then at 4 days. The possible role of these lipids in the cytopathic effects of HIV-infection is discussed. In addition, an improved methodology to quantitate simultaneously diacylglycerol and alkylacylglycerol in crude lipid extracts, based upon their phosphorylation by E. coli diacylglycerol kinase, is presented.