Significant amounts of H2O2 were produced when the polyamines, spermine, or spermidine were incubated with a Krebs-Ringer phosphate buffer that contained bovine serum albumin (Fraction V). This effect was specific for certain amines and could be prevented by treatment of the albumin fraction with isoniazid or aminoguanidine. These features suggest that H2O2 is formed during oxidative deamination of the polyamines catalized by spermine oxidase, a known contaminant of Fraction V bovine serum albumin. The insulin-like effects elicited by polyamines in fat cells (e.g. enhancement of glucose transport and inhibition of cAMP-mediated lipolysis) were dependent on H2O2 production. Incubation of cells with catalase or treatment of the albumin fraction with isoniazid abolished the stimulation of glucose uptake by polyamines but did not alter the stimulatory effects of insulin or vitamin K5. The H2O2 generating activity was partially separated from the albumin by gel filtration; only those fractions which formed H2O2 provided support for the activation of glucose transport by polyamines. Also, the time needed to activate glucose uptake was markedly shortened by incubation of the albumin buffer with the polyamines before addition of the cells. These findings indicate that the polyamines do not themselves mimic the actions of insulin but that the insulin-like effects result from the formation of H2O2 which has been shown to stimulate glucose transport.