How fructose 2,6-bisphosphate and metabolic intermediates interact to regulate the activity of the cytosolic fructose 1,6-bisphosphatase in vitro has been investigated. Mg(2+) is required as an activator. There is a wide pH optimum, especially at high Mg(2+). The substrate dependence is not markedly pH dependent. High concentrations of Mg(2+) and fructose 1,6-bisphosphate are inhibitory, especially at higher pH. Fructose 2,6-bisphosphate inhibits over a wide range of pH values. It acts by lowering the maximal activity and lowering the affinity for fructose 1,6-bisphosphate, for which sigmoidal saturation kinetics are induced, but the Mg(2+) dependence is not markedly altered. On its own, adenosine monophosphate inhibits competitively to Mg(2+) and noncompetitively to fructose 1,6-bisphosphate. In the presence of fructose 2,6-bisphosphate, adenosine monophosphate inhibits in a fructose 1,6-bisphosphate-dependent manner. In the presence of adenosine monophosphate, fructose 2,6-bisphosphate inhibits in Mg(2+)-dependent manner. Fructose 6-phosphate and phosphate both inhibit competitively to fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate does not affect the inhibition by phosphate, but weakens inhibition by fructose 6-phosphate. Dihydroxyacetone phosphate and hydroxypyruvate inhibit noncompetitively to fructose 1,6-bisphosphate and to Mg(2+), but both act as activators in the presence of fructose 2,6-bisphosphate by decreasing the S(0.5) for fructose 1,6-bisphosphate. A model is proposed to account for the interaction between these effectors.