Characteristics of fatty acylation of myelin proteolipid protein (PLP) in vitro were compared with the corresponding process in vivo. Rapid and efficient separation of labelled PLP from other proteins and lipids was effected by extraction into chloroform/methanol/0.1 N HCl (10/10/1) and chromatography on Sephadex LH-60 in the same solvent. Covalent linkage of [3H]-palmitate to PLP was demonstrated by repetitive chromatography on LH-60, thin layer chromatography, and polyacrylamide gel electrophoresis. Reductive cleavage with sodium borohydride of PLP acylated in vitro or in vivo yielded [3H]-hexadecanol, identifying at least one of the acyl linkages as a thiolester bond. When PLP was acylated with acyl-CoA as the fatty acid donor, the reaction occurred non-enzymatically as supported by the following observations: 1) acylation activity increased with increasing pH above pH 7.5, 2) acylation activity was heat stable, 3) acylation activity was not removed from PLP during purification in organic solvents or in Triton X-100-containing buffers, and 4) acylation of tryptic fragments occurred in the absence of an exogenously added enzyme source. The relevance of in vitro fatty acylation of PLP to that in vivo was confirmed by comparison of proteolytically derived peptide maps that showed that likely the same domain of PLP was acylated in vitro and in vivo.