The lipid, sterol, and fatty acid compositions of Heterorhabditis bacteriophora infective juveniles reared by in vivo and in vitro methods (Galleria mellonella, Popillia japonica, solid and liquid culture) were compared. The greatest accumulation of lipids per dry weight was achieved by growing nematodes in P. japonica and solid culture (54.7 and 60.3%, respectively). Nematodes produced by liquid fermentation accumulated lower levels of sterols (2%) per total lipids compared with other production methods. However, the level of polar lipids was higher in nematodes produced in artificial media. Fractionation of the polar lipids revealed that an increased level of phosphatidylglycerol accompanied production in liquid culture. This was not observed for nematodes produced in solid culture in which the major polar lipid fractions were phosphatidylcholine and phosphatidylethanolamine. Lipids from nematodes produced in artificial media had more linoleic (C18: 2) fatty acids, whereas lipids from in vivo cultures had more stearic (C18: 0) fatty acids. The ratio of saturated to unsaturated fatty acid composition decreased in cultures produced in vitro. Artificial media composition should be adjusted to meet the nutritional composition of a natural host. Media for H. bacteriophora culture should be modified to boost deficiencies in sterols (e.g., liver extract) and oleic acid (e.g., olive oil). r 1999 Academic Press