In February to March, wild brown trout Salmo trutta were captured by electrofishing in a natural watercourse (tributaries of the River Lille Aa, Denmark), individually tagged (Passive Integrated Transponders), and released. Representatives of the tagged brown trout were recaptured on the release sites in April by electrofishing and eventually caught in downstream smolt traps (‘migrants’) placed in the main river or by electrofishing (‘residents’) on the initial sites in June. Upon each capture, smolt appearance and body size were evaluated, and a non-lethal gill biopsy was taken and used for Na+,K+-ATPase analysis. Based on repetitive gill enzyme analysis in individual fish, a retrospective analysis of the rate of development in individual brown trout ultimately classified as migrants or residents was performed. Two months prior to migration, a bimodal morphological and physiological (gill Na+,K+-ATPase) development concurred and was related to the subsequent differentiation into resident and migratory fractions of each population. This differentiation was unrelated to growth rate and body size of individual fish but skewed in favour of migratory females. Individuals destined to become migrants developed a smolt-like appearance before the onset of migration and had higher rate of change of gill Na+,K+-ATPase activity than fish remaining residents. The rate of change of gill Na+,K+-ATPase activity was independent of the distance migrated to the trap (3–28 km). Thus in bimodal wild brown trout populations a major increase in enzyme activity takes place before migration is initiated and is a characteristic of migratory individuals only.