An acylation/deacylation cycle is required to maintain the steady state subcellular distribution and biological activity of S-acylated proteins. The cycle steps are catalyzed by protein acyl tranferases (PATs) and acyl-protein thioesterases (APTs), respectively. PATs has been recently identified and substrates are being characterized. In contrast, proteins and mechanisms involved in protein deacylation are poorly understood. We studied in CHO-K1 and HeLa cells the deacylation step of the full-length (GAP-43^full) or the acylation motif (^N13GAP-43) of diacylated GAP-43 fused to YFP as well as their single acylated mutants GAP-43^full(C3S) or ^N13GAP-43(C3S). Using biochemical assays and live cell fluorescence microscopy, it was demonstrated that single acylated mutants, but not dually acylated GAP-43, were deacylated by treatment with an inhibitor of protein palmitoylation. The ectopic expression of APT-1, the unique so far described cytosolic APT, did not affect the acylation state of dually acylated ^N13GAP-43 but increased the deacylation rate of the single acylated protein. Surprisingly, APT1 transcripts were detected by RT-PCR in HeLa cells, but not in CHO-K1 cells where deacylation activity was also demonstrated. These results suggest the presence of additional enzymes with acyl-protein thioesterase activity involved in the deacylation of GAP-43 in CHO-K1 cells.