Because serotonin (5-HT) is a neurotransmitter associated with circadian rhythm regulation, we explored a possible relation among 5-HT, serotonin metabolite, 5-hydroxyindolacetic acid (5HIAA), and the functional polymorphism of the serotonin transporter gene (SLC6A4) promoter with rotating shift work.683 men were included in this study: 437 day workers were compared with 246 rotating shift workers.Platelet 5-HT content differed significantly (P = 0.002) between day workers (41.28+/-1.99 pg/mg) and rotating shift workers (37.91+/-4.16 pg/mg); 5-HIAA content was also significantly (P = 0.00004) higher in day workers (11.40+/-0.82 pg/mg) than in rotating shift workers (9.33+/-1.02 pg/ mg). We looked for further differences in SLC6A4 promoter (5-HTTLPR, 44 bp insertion: long (L)/deletion: short (S) alleles). We found a significant (P = 0.016) difference in genotype distribution between day workers LL: 126 (28.8%), LS: 202 (46.2%), and SS: 109 (24.9%), and rotating shift workers LL: 47 (19.1%), LS: 124 (50.4%), and SS: 75 (30.5%). When we divided the subjects between workers with less and more than 60 month rotating shift-work exposure, the difference in SLC6A4 genotypes frequency was only significant in the group with > or =60 months (P = 0.011). In addition, there was a significantly lower content of platelet 5-HIAA in S allele carriers in comparison with the other genotypes (SS: 9.2+/-1.0 pg/mg vs. SL/LL: 11.0+/-0.8 pg/mg, P <0.02).Platelet 5-HT and 5-HIAA contents were significantly lower in rotating shift workers than day workers, and there was a significant association between the S variant of SLC6A4 promoter and shift work. These findings may be important for targeting effective therapeutic strategies to ameliorate the associated comorbidities and behavioral problems in rotating shift workers.