Tryptophan depletion is a widely used paradigm to study serotonin system-related mechanisms in the pathophysiology and treatment of depression. There is convincing evidence that tryptophan depletion primarily and selectively affects serotonergic transmission. The behavioral data in healthy controls with and without genetic risk for depression, and in patient populations during the symptomatic phase of depression and when being remitted, suggest a trait abnormality of serotonin function in depression and that antidepressants may compensate for the underlying deficit. Tryptophan depletion may be a useful tool to create more integrative models for the pathophysiology of depression that take into account neurobiological systems beyond monoamines. More recent studies combining tryptophan depletion with genetic variables may provide an important approach for studying gene/environment interactions using candidate genes to define endophenotypes, which ultimately will improve currently used diagnostic categories and help to generate more advanced models to understand the neurobiology of depression. This may lead to the development of truly novel treatment approaches for depression.