Regulatory effects which adenine nucleotides are known to exert on glycolysis and gluconeogenesis have been demonstrated in the isolated perfused rat kidney and in kidney cortex slices. These preparations rapidly converted added ATP into ADP and AMP; added ADP was also converted into AMP and added AMP was rapidly dephosphorylated. AMP produced from ATP was dephosphorylated at a much slower rate than added AMP, especially when the initial ATP concentration was high (10 mM). Glucose synthesis from propionate (2.5 mM) or lactate (10 mM) was inhibited by adding adenine nucleotides to the incubation and perfusion media. In contrast, the oxygen consumption and substrate utilization were not significantly inhibited. Inhibitions of gluconeogenesis caused by the addition of ATP and ADP were proportional to the AMP concentration generated. Glucose uptake was stimulated 30–50% by added ATP or AMP, but the extra glucose removed was not oxidized. Three-carbon phosphorylated intermediates, especially α-glycerophosphate, accumulated in the tissue. The effects of added adenine nucleotides are mainly due to AMP, which inhibits fructose disphosphatase and deinhibits phosphofructokinase. ATP or ADP (10 mM) added to the medium perfusing an isolated rat kidney increased the renal vascular resistance and diminished the flow rate for several minutes. The effect of AMP (10 mM) was less marked and adenosine (10 mM) had no effect.