Several proteins involved in transmembrane signaling have been shown previously to be modified covalently by long-chain fatty acids. Using the BC3H1 cell line, which contains a broad array of fatty acylated proteins, we have examined the possibility that acylation of certain proteins is modulated in response to mitogenic stimulation. In the present study, we describe a 64-kDa palmitoylated protein, referred to as p64, that is deacylated following stimulation of quiescent cells with fetal bovine serum, fibroblast growth factor, and phorbol dibutyrate. Western blot analysis of membrane and soluble fractions using a polyclonal antibody against p64 revealed that approximately 70% of p64 in unstimulated cells is present in the cytosol in a non-acylated form, whereas palmitoylated p64 is found exclusively in the membrane fraction. Extraction of membranes with 0.5 M sodium chloride, 0.2 M sodium pyrophosphate, or 0.2 M sodium carbonate failed to release p64, suggesting that the acylated form of this protein is tightly associated with membranes. Pulse labeling of proteins in quiescent cells with [3H] palmitate and subsequent chasing in medium containing 20% fetal bovine serum, fibroblast growth factor, or phorbol dibutyrate revealed that the fatty acid associated with p64 undergoes mitogen-stimulated turnover, whereas turnover of fatty-acid on other acylated proteins is not observed. Palmitate is the predominant fatty acid associated with p64; however, small amounts of covalent myristate are also detected. Both fatty acids are attached post-translationally to p64 through a hydroxylamine-sensitive linkage, suggesting that acylation of this protein is catalyzed by a palmitoyl transferase with relaxed specificity for fatty acid substrates. Together, these results suggest that palmitoylation may participate in the association of p64 with the plasma membrane and that mitogen-dependent deacylation might alter interactions between this protein and other membrane components.