Chemical and enzymatic methods have been developed to measure small quantities (10(-8) - 10(-10) mol) of acyldihydroxyacetone phosphate in animal tissues. Lipids extracted from tissue samples with acidic CHCl3/methanol were subjected to solvent partitioning at two different pH values for partial purification of this keto-lipid from other lipids. This lipid was then estimated radiometrically either by chemical reduction with NaB3H4 or by enzymatic reduction with [4B-3H]NADPH using a partially purified acyldihydroxyacetone-phosphate reductase (EC 1.1.1.101). Thin-layer chromatography revealed the presence of a number of 3H-labeled lipids in the NaB3H4-reduced product and further purification of the product was necessary to estimate the amount of acyl[2-3H]glycerol 3-phosphate formed. The enzymatic reduction was very specific for acyl/alkyldihydroxyacetone phosphate. The amounts (nmol/g) of these keto-lipids estimated in different tissues by the enzymatic method were 10.06 +/- 0.64 (guinea pig liver), 4.3 +/- 0.15 (rat liver), 2.1 (rat testis), 1.5 (rad kidney) and 1.2 (rat brain). Monoacylglycerol 3-phosphate, i.e., lysophosphatidic acid, which was co-purified with acyldihydroxyacetone phosphate, was found to be present in relatively larger amounts in tissues. The amounts (nmol/g) of this lipid, estimated by enzymatically measuring the amounts of sn-glycerol 3-phosphate released after alkaline methanolysis of the partially purified lipid extracts, were 143 (guinea pig liver), 58 (rat liver), 53 (rat kidney) and 92 (rat brain). Stearic acid (18:0) was found to be the major (65%) fatty acid present in the lysophosphatidate purified from guinea pig liver.