Using [14C]palmitoyl-CoA as donor and deacylated (fatty acid-free) structural proteins of Semliki Forest virus as exogenous acceptors, palmitic acid was incorporated into polypeptide in a cell-free system with microsomes of baby hamster kidney cells, chicken embryo fibroblasts, and rat liver cells. Out of the four viral proteins (E1, E2, E3, and C) only E1 becomes acylated enzymatically. The protein bound fatty acids of the in vitro product are resistant to detergents and to organic extractions but can be released with hydroxylamine thus affording the typical features of acyl-proteins. Fatty acid transfer to E1 requires the presence of microsomes and is abolished when microsomal membranes are omitted or boiled prior to the incubation. Exogenous acceptor protein E1 had to be deacylated prior to the incubation in order to function during acylation in vitro since no fatty acid chains were transferred into untreated viral E1. Acylation of E1 is time- and temperature-dependent and can be stimulated by increasing the concentrations of acceptor protein, microsomal membranes, or of exogenous fatty acid donor. While Mg2+ does not influence the transfer reaction, Mn2+ leads to a dose-dependent inhibition commencing at a concentration of 1 mM. The cell-free acylation activity fails to show high specificity with regard to the chain length or degree of saturation of the acyl chain used as lipid substrate since palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and myristic acid (C14:0) are all transferred onto E1 as long as ATP is present in the incubation mixture. The type of detergent and its concentration were found to be critical for this acylation in vitro. Nonidet P-40 or Triton X-100 both up to a 0.2% concentration allowed the reaction while no enzymatic transfer of palmitic acid onto E1 was detected in the presence of octyl-beta-D-glucoside and Tween 20 at the conditions used for the in vitro incubation. However, when acyl transfer onto lipid acceptors was monitored, the incorporation of fatty acids into the neutral- and phospholipids functioned normally in octyl-beta-D-glucoside only, while Nonidet P-40 and Triton X-100 inhibited the acylation of all neutral lipids and of most of the phospholipids completely.(ABSTRACT TRUNCATED AT 400 WORDS)