In the post‐microsomal supernatant of pancreatic islets, prepared from fasted or fed rats, D‐fructose 1‐phosphate increased the activity of glucokinase by 20–30% as measured in the presence of D‐glucose 6‐phosphate and D‐fructose 6‐phosphate. Such an activation was less marked than that found in liver extracts. The islet cytosol was also found to inhibit purified liver glucokinase, and this effect was antagonized by D‐fructose 1‐phosphate. In the presence of hexose 6‐phosphates, partially purified islet glucokinase was inhibited by the hepatic glucokinase regulatory protein in a D‐fructose‐1‐phosphate‐sensitive manner. In intact islets, D‐glyceraldehyde stimulated the generation of ¹⁴C‐labelled D‐fructose 1‐phosphate from D‐[U‐¹⁴C]glucose and increased the production of ³H₂O from D‐[5‐³H]glucose. These findings suggest that the activity of glucokinase in islet cells may be regulated by a protein mediating the antagonistic effects of D‐fructose 6‐phosphate and D‐fructose 1‐phosphate in a manner qualitatively similar to that operating in hepatocytes, but with lower efficiency.