4-Hydroxynonenal binds rapidly to Na(+)-K(+)-ATPase, and this was accompanied by a decrease in measurable sulfhydryl groups and a loss of enzyme activity. The I50 value for Na(+)-K(+)-ATPase inhibition by 4-hydroxynonenal was found to be 120 microM. Although the sulfhydryl groups could be completely restored with beta-mercaptoethanol during the reaction of the Na(+)-K(+)-ATPase-HNE-adduct, the Na(+)-K(+)-ATPase activity was only partially restored by this reducing agent. A combination of hydroxylamine and beta-mercaptoethanol yielded the greatest recovery of enzyme activity, 85% of original. Thus, 4-hydroxynonenal binding to Na(+)-K(+)-ATPase led to an irreversible decrease of enzyme activity under the conditions employed. It is hypothesized that 4-hydroxynonenal reacts with sulfhydryls at sites on the enzyme that are inaccessible by beta-mercaptoethanol. Furthermore, evidence was obtained that 4-hydroxynonenal reacts with other amino acids such as lysine to form adducts that also interfere with protein function.