We studied the antidepressant-like effect of paroxetine in strains of mice carrying different isoforms of tryptophan hydroxylase-2 (TPH-2), the enzyme responsible for the synthesis of brain serotonin (5-HT). The effect of paroxetine alone and in combination with pharmacological treatments enhancing or lowering 5-HT synthesis or melatonin was assessed in the forced swimming test in mice carrying allelic variants of TPH-2 (1473C in C57BL/6 and 1473G in DBA/2 and BALB/c). Changes in brain 5-hydroxytryptophan (5-HTP) accumulation and melatonin levels were measured by high-performance liquid chromatography. Paroxetine (2.5 and 5 mg/kg) reduced immobility time in C57BL/6J and C57BL/6N mice but had no such effect in DBA/2J, DBA/2N and BALB/c mice, even at 10 mg/kg. Enhancing 5-HT synthesis with tryptophan reinstated the antidepressant-like effect of paroxetine in DBA/2J, DBA/2N and BALB/c mice whereas inhibition of 5-HT synthesis prevented the effect of paroxetine in C57BL/6N mice. The response to paroxetine was not associated with changes in locomotor activity, brain melatonin or brain levels of the drug measured at the end of the behavioral test. These results support the importance of 5-HT synthesis in the response to SSRIs and suggest that melatonin does not contribute to the ability of tryptophan to rescue the antidepressant-like effect of paroxetine.