Technical Abstract: The lipid profiles of cotton fiber cells were determined from total lipid extracts of elongating and maturing cotton fiber cells to see if the membrane lipid composition changed during the phases of rapid cell elongation or secondary cell wall thickening. Total fatty acid content increased during elongation and decreased, thereafter, likely reflecting the assembly of the expanding cell membranes during elongation and the shift to membrane maintenance (and increase in secondary cell wall content) in maturing fibers. Analysis of lipid extracts by electrospray ionization and tandem mass spectrometry (ESI-MS/MS) revealed that in elongating fiber cells (7-10 days post anthesis), the polar lipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) phosphatidylglycerol (PG) were most abundant. These same glycerolipids were found in similar proportions in maturing fiber cells (21 dpa). Detailed molecular species profiles were determined by ESI-MS/MS for all glycerolipid classes, and ESI-MS/MS results were consistent with lipid profiles determined by HPLC and evaporative light scattering detection (ELSD). The predominant molecular species of PC,PE, PI and PA was 34:3 (16:0, 18:3), while 36:6 (18:3,18:3), also was prevalent. Total fatty acid analysis of cotton lipids confirmed that indeed linolenic (18:3) and palmitic (16:0) acids were the most abundant fatty acids in these cell types. Bioinformatics data were mined from cotton fiber EST databases in an attempt to reconcile expression of lipid metabolic enzymes with lipid metabolite data. Together these data form a foundation for future studies of the functional contribution of lipid metabolism to the development of this unusual and economically important cell type.