Phospholipase A1 activities have been detected in most cells where they have been sought and yet their characterization lags far behind that of the phospholipases A2, C and D. The study presented here details the first cloning and characterization of a cytosolic PLA1 that exhibits preference for phosphatidylcholine (GPCho) substrates. Trypanosoma brucei phospholipase A1 (TbPLA1) is unique from previously identified eukaryotic PLA1 because it is evolutionarily related to bacterial secreted PLA1. A T. brucei ancestor most likely acquired the PLA1 from a horizontal gene transfer of a PLA1 from Sodalis glossinidius, a bacterial endosymbiont of tsetse flies. Nano-electrospray ionization tandem mass spectrometry analysis of TbPLA1 mutants established that the enzyme functions in vivo to synthesize lysoGPCho metabolites containing long-chain mostly polyunsaturated and highly unsaturated fatty acids. Analysis of purified mutated recombinant forms of TbPLA1 revealed that this enzyme is a serine hydrolase whose catalytic mechanism involves a triad consisting of the amino acid residues Ser-131, His-234 and Asp-183. The TbPLA1 homozygous null mutants generated here constitute the only PLA1 double knockouts from any organism.