Six ethanolamine sphingophospholipids from axenically cultured Paramecium tetraurelia were isolated from cells and purified ciliary fractions, and were characterized. The sphingolipids comprised 10.7% of whole cell and 32.5% of ciliary ethanolamine phospholipid fractions purified by ion exchange column chromatography. The individual sphingolipids were characterized by thin-layer chromatographic analyses of parent compounds and the polar head group and long chain base moieties, gas-liquid chromatography, and mass spectrometry of amide-linked fatty acids and long chain bases, and nuclear magnetic resonance of the compounds. Colorimetric assays of differential hydrolysis products and 31P nuclear magnetic resonance were used to determine the nature of phosphorus linkages. The sphingolipids were identified as N-acyl-trans-4-hydroxy-sphinganine-1- phosphonoethanolamine , N-acyl-trans-4-hydroxy-sphinganine-1-phosphoethanolamine, N-acyl-sphingenine-1- phosphonoethanolamine , N-acyl-sphingenine-1-phosphoethanolamine, N-acyl-sphinganine-1- phosphonoethanolamine and N-acyl-sphinganine-1-phosphoethanolamine. All six had greater than 90% saturated fatty acids. These sphingolipids were quantified by radioisotope methods and plate densitometry of thin-layer chromatograms. Changes in the relative amounts of each species were detected in cells grown in different culture media as well as in cells at different culture ages.