We characterized the degree of plasticity in thermal tolerance (assessed as critical thermal maxima; CTMax) and the relationship between thermal tolerance and underlying physiological and biochemical factors in two subspecies of a teleost fish, Fundulus heteroclitus. CTMax was not affected by repeated daily heat shock, but increased within a few days in response to warm acclimation. Loss of tolerance with acclimation to lowered temperatures occurred more slowly. Exposure to hypoxia decreased CTMax, and hyperoxia had no effect. CTMax showed a daily rhythm in both subspecies. Thermal acclimation changed the value of CTMax but did not affect the amplitude of the rhythm. Exposure to altered photoperiod had complex effects with a summer photoperiod producing a daily rhythm at higher CTMax than a spring photoperiod, and a winter photoperiod removing the rhythm. There was no daily rhythm in routine metabolic rate in either subspecies. There was no relationship between CTMax and the protein levels of the constitutive 70 and 90 kDa heat shock proteins (HSC70, HSP90β) in gill, or with mRNA levels of hsc70 in liver. There was a daily rhythm in the basal levels of the inducible hsp70-2 mRNA. Induction of hsp70-2 mRNA with mild heat shock occurred only in the evening and at night, and not during the day. These results demonstrate that there is substantial plasticity of thermal tolerance in killifish, and that this plasticity does not differ between subspecies. CTMax has a complex relationship with physiological and biochemical mechanisms that have been hypothesized to affect thermal tolerance.