The serotonin (5-HT) system is implicated in incentive motivational processes. The present study utilized the acute tryptophan depletion (ATD) procedure to investigate the effect of temporarily lowering 5-HT synthesis on motivation in healthy volunteers, stratifying the results by allelic variation at the serotonin transporter gene (5-HTTLPR). ATD resulted in a robust reduction in plasma tryptophan concentration. Consistent with a previous study, ATD attenuated motivationally speeded action on the Cued-Reinforcement Reaction Time task. The present investigation revealed that this effect was restricted to volunteers of the ss genotype, whereas ll volunteers exhibited intact motivationally speeded action following ATD (treatment x reinforcement probability x genotype interaction: F1,26=5.8, p=0.024). Furthermore, tryptophan availability to the brain was correlated positively with motivationally speeded action following ATD in the ss genotype group (rho13=0.71, p=0.006), whereas this correlation was negative in the ll genotype group (rho14=-0.60, p=0.023). This is the first study to suggest that allelic variation at the 5-HTTLPR mediates motivational responses to ATD in healthy volunteers. These data indicate that the s allele at the 5-HTTLPR may confer risk for depression via its effect on incentive motivational processing, and highlight the importance of genetic variation in determining individual responses to pharmacological treatments.