Separate groups of banded killifish, Fundulus diaphanus, were maintained on a LD 12:12 photoregime at either 5° or 20°C for a total duration of 16 days. Following this acclimation period, blood samples were taken by cardiac puncture at six different times of day and analyzed for serum cortisol by radioimmunoassay. Fish acclimated to 20°C had a unimodal daily pattern of serum cortisol with the peak concentration occurring during the four hour period before the onset of light. The daily pattern of cortisol of the fish acclimated to 5°C was bimodal with peak cortisol values measured at the onset and the offset of light. These thermally induced changes in cortisol hormone patterns may be related to other temperature dependent seasonal changes such as the annual cycle of reproductive development or changes in the time of daily locomotor activity.