Background: Many recent studies of serotonin transporter gene by environment effects predicting depression have used stress assessments with undefined or poor psychometric methods, possibly contributing to wide variation in findings. The present study attempted to distinguish between effects of acute and chronic stress to predict depressive symptoms at age 20 among 346 youth varying in polymorphisms of the 5HTT gene who had been assessed at ages 15 and 20. Methods: Interview measures assessed major acute life events between 15 and 19, and multiple interviews and questionnaires with youths and their parents at youth age 15 provided an index of chronic family stress. Lg alleles were reclassified as S. Results: Chronic family stress at age 15 predicted higher depression scores at 20 among those with one or two S alleles, and the effects of genetic moderation were significant only for females. Gene-environment interactions with acute stress were nonsignificant. Conclusions: Careful measurement and separation of the effects of chronic and acute stress, and gender, are encouraged in the study of mechanisms of the stress-depression association.