Context: Diet reportedly alters serum sex hormone concentrations in adults, but little is known about the influence of diet during puberty on these hormones. Objective: We aimed to determine if an intervention to lower fat intake during adolescence alters serum sex hormone concentrations and progression through puberty. Design: In 1990-1997, we conducted an ancillary study to the Dietary Intervention Study in Children (DISC), a multicenter randomized controlled clinical trial to test safety and efficacy of a cholesterol-lowering dietary intervention in children. Participants: Healthy, prepubertal, 8-10 yr olds with elevated LDL-cholesterol were randomized to usual care or a behavioral intervention. Of 362 randomized DISC boys, 354 participated in the ancillary study. 84% of boys attended last visits when their median time on trial was 7.1 yr. Intervention: The behavioral intervention continued throughout the duration of the trial and promoted a diet with 28% energy from total fat, <8% from saturated fat, 9% from polyunsaturated fat, and <75 mg cholesterol/1000 kcal. Outcome Measures: The main outcome measure for boys formulated before study initiation was non-SHBG bound testosterone concentration. Secondary outcomes included serum total testosterone, dihydrotestosterone, androstenedione, estradiol, estrone, and SHBG, and Tanner stage. Results: There were no significant treatment group differences in boys' serum hormone levels, SHBG or Tanner stages at any individual visit or over the course of the trial when evaluated by longitudinal models. Conclusion: Modest reductions in total fat, saturated fat, and possibly energy intake do not alter progression through puberty or serum sex hormone concentrations in adolescent boys.