The inner opercular epithelium of the killifish, Fundulus heteroclitus, may be separated as a single epithelial sheet and mounted in an Ussing-style chamber with the short-circuit current (Isc) approximating chloride secretion. Steady state opercular Isc values in seawater-adapted killifish were higher in tissues removed from fish in the afternoon than those observed in the morning. Opercular conductance was also higher in the afternoon than the morning. The opercular steady state Isc from killifish on a 13:11 light:dark cycle exhibited a diel variation with the peak in the middle of the light cycle and a nadir near the end of the dark phase. Temperature and salinity remained constant during the experimental period. Daily variation cued by the photoperiod should be considered when using this preparation as an experimental model for ion transport in the gill of a seawater-adapted fish.