Objective We assessed serum homocysteine (tHcy) and folate concentrations among US adolescents before and after fortification of cereal-grain products with folic acid, and associations with demographic, behavioural and physiological factors. Design Observational study conducted among participants of a randomized trial. Setting The Child and Adolescent Trial for Cardiovascular Health (CATCH) study. Subjects Adolescents (n 2445) in grades 8 (pre-fortification, mean age 14 years) and 12 (post-fortification, mean age 18 years). Results Average serum concentrations of tHcy, folate and vitamin B6 increased by 17 %, 16 % and 14 %, respectively, while serum concentrations of vitamin B12 decreased by 11 % post-fortification. Folic acid fortification provided, on average, an additional intake of 118 μg folate/d. Male sex (P < 0·0001) and white race (P = 0·0008) were associated with significantly greater increases in tHcy concentration, while increases in BMI (P = 0·006) and serum folate concentration (P < 0·0001) were associated with significant decreases in tHcy concentration. Female sex (P < 0·0001), non-smoking (P < 0·0001), use of multivitamins (P < 0·0001) and higher dietary intake of folate (P = 0·001) were associated with significantly greater increases in serum folate concentrations. From grade 8 to grade 12, the upward age trend in serum tHcy concentration was uninterrupted in its course (P > 0·50); whereas serum folic acid concentration showed a downward trend that incurred a discrete jump upward (17 % higher; P < 0·0001) with fortification. These trends differed significantly for males v. females (P < 0·001 for interaction). Conclusions Fortification had a significant impact on improving folate status but not serum tHcy concentrations among US adolescents.