Large changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) in the pineal gland control the rhythmic production of the time-keeping hormone melatonin. The activity of AANAT reflects changes in the amount and activation state of the AANAT protein, both of which increase at night. The molecular basis of this regulation is now becoming known, and recent data indicate that this involves phosphorylation-dependent binding to the 14-3-3 protein at two sites, one of which, Ser-205, is located several residues from the C terminus. In this study, we determined whether substitution of this residue with a non-hydrolyzable the phosphoserine/phosphothreonine mimetic would promote binding to the 14-3-3 protein and enhance cellular stability. To accomplish this, a C-terminal AANAT peptide containing the phosphonodifluoromethylene alanine at Ser-205 was synthesized and fused to bacterially expressed AANAT(30-199) using expressed protein ligation. The resulting semisynthetic protein has enhanced affinity for the expressed 14-3-3 protein and exhibits greater cellular stability in microinjection experiments, as compared with the unmodified AANAT. Enhanced 14-3-3 binding was also observed using humanized ovine AANAT, which has a different C-terminal sequence (Gly-Cys) than the ovine enzyme (Asp-Arg), indicating that that characteristic is not unique to the ovine enzyme. These studies provide the first evidence that substitution of Ser-205 with the stable phosphomimetic amino acid phosphonodifluoromethylene alanine enhances binding to 14-3-3 and the cellular stability of AANAT and are consistent with the view that Ser-205 phosphorylation plays a critical role in the regulation of AANAT activity and melatonin production.