SummarySubstrate control of HK-II activity was studied in rat adipose tissue incubated in vitro for 18 hr in a modified TC-199 medium. In addition to glucose, the metabolizable substrates of the enzyme, fructose and mannose, maintained higher levels of HK-II than did control incubations. 2DG and xylose, both nonmetabolizable sugars which compete with glucose for binding to HK, decreased enzyme activity. Hex-oses which do not bind to the active site of the enzyme, 3OMG and galactose, have minimal effect on enzyme activity. Nonhex-ose energy sources did not maintain high HK-II activity. G6P, the product of glucose phosphorylation by HK, increased HK-II despite its minimal transport into the cell. It is suggested that G6P may mediate the effects of sugars and insulin on HK-II.