Metallothionein (MT), a cysteine-rich metal binding protein, is considered to play an essential role in the regulation of intracellular metals. Induction of MT in mammalian and nonmammalian tissues following heavy metal exposure may serve as a defense mechanism and a biomarker of environmental exposure to chemical stressors such as toxic metals. In this study, MT messenger RNA (mRNA) expression was characterized in male and female nonspawning and spawning killifish (Fundulus heteroclitus) following an 8-day exposure to specific sublethal stressors, which included temperature perturbation (26°C or 10°C) and/or 6 ppb of waterborne cadmium chloride (CdCl2). Hepatic, gill, and intestinal MT mRNA, expressed as copy number per microgram of total RNA, was assessed by reverse transcriptase–polymerase chain reaction and electrochemiluminescence using winter flounder (Pleuronectes americanus) MT complementary DNA primers. Liver, gill, and intestine MT mRNA expression was significantly (P < 0.05) increased in nonspawning killifish exposed to 26°C compared with those exposed to 19°C (control). In addition, a significant (P < 0.05) increase in gill MT mRNA induction was observed in nonspawning killifish exposed to 6 ppb of waterborne CdCl2 compared with controls. The results of this study demonstrate significant MT mRNA induction in nonspawning killifish following short-term exposure to physiological and chemical stressors. Thus, further research may be necessary before the use of killifish MT mRNA induction as a biomarker of environmental chemical stress exposure alone.