The purpose of this study was to test hypotheses regarding (1) relations among negative affect and hormones of gonadal and adrenal origin in young adolescents, at three times of measurement, over a one-year period; and (2) stability of negative affect. The sample consisted of 10- to 14-year-old boys (N=56) and 9- to 14-year-old girls (N=52). The adolescents were assessed three times at 6-month intervals over one year. Serum levels of gonadotropins, gonadal steroids, adrenal androgens, and cortisol were assessed, as well as stage of pubertal development (Tanner criteria). The negative affect assessments consisted of self-report questionnaire and interview measures of anxiety and depressive affect, as well as mother reports of internalizing behavior problems. In the concurrent (cross-sectional) analyses, boys reporting higher levels of negative affect tended to be those at higher genital stage or older age, with lower testosterone and cortisol levels and lower dehydroepian-drosterone sulphate levels. In the longitudinal analyses, negative affect, and to a lesser extent hormone levels at the first time of measurement predicted negative affect 12 months later. The findings suggest that puberty-related hormone levels should be considered along with psychological characteristics in examining the processes involved in the development of negative affect during the pubertal years.