Isozymes of glutathione reductase (GR) have been purified from red spruce (Picea rubens Sarg.) needles. Two isozymes could be separated by anion-exchange chromatography from both nonhardened or cold-hardened tissue. Based on chromatographic elution profiles, the isozymes were designated GR-1NH and GR-2NH in preparations from nonhardened needles, and GR-1H and GR-2H in preparations from hardened needles. N-terminal sequencing and immunological data with antisera obtained against GR-1H and GR-2H established that the isozymes from hardened needles are different gene products and show significant structural differences from each other. Chromatographic, electrophoretic, and immunological data revealed only minor differences between GR-2NH and GR-2H, and it is concluded that these isozymes are very similar or identical. Anion-exchange chromatography and native polyacrylamide gel electrophoresis also established that GR-1NH and GR-1H are different proteins. From these data we conclude that GR-1H is a distinct gene product, present only in hardened needles. Therefore, GR-1H can be considered to be a cold-hardiness-specific GR isozyme, and GR-1NH can be considered to be specific for nonhardened needles. It is proposed that GR-1H is a cold-acclimation protein.