Evidence is presented for the simultaneous release of platelet-activating factor (PAF-acether) and of its deacetylated derivative (lyso-PAF-acether) from hog leukocytes. On the basis of spectroscopy and chemical reactions, the structure of O-deacetyl-PAF is shown to be 1-O-alkylglyceryl 3-phosphorylcholine, an alkyl ether analog of lyso-phosphatidylcholine. Acetylation of lyso-PAF yields a compound with biological activity and chromatographical behavior indistinguishable from those of native PAF. Lyso-PAF may be considered to be either the precursor or the enzymatic degradation product of PAF. The usefulness of chemical ionization mass spectrometry for structural determination of phospholipids is also demonstrated.