Insulin binding and insulin stimulated amino acid and glucose uptake were determined in cultured HTC hepatoma cells in the presence of Ca2+ and ruthenium red (RR) in order to further characterise the putative calcium binding site on the receptor. These ions increased insulin receptor high affinity binding and the sensitivity of these responses to insulin. The insulin concentration required to half-maximally stimulate amino acid uptake decreased significantly from 26.9 +/- 5.8 ng/ml to 6.0 +/- 1.3 ng/ml in the presence of 10 mM Ca2+ and to 1.3 +/- 0.5 ng/ml in the presence of RR. The effect of Ca2+ and RR was more pronounced on insulin stimulated glucose uptake. These agents also increased receptor-effector coupling, reducing the percentage of occupied receptors required for maximal insulin stimulation of amino acid uptake from 10.8% in control cells to 3.4 and 1.4% in the presence of Ca2+ and RR respectively. The receptor occupancy required to produce maximal insulin responses on glucose uptake decreased from 20% (control) to 3.8% (Ca2+ and RR). We hypothesize that since Ca2+ and RR have similar effects, that occupation of Ca2+ binding sites on the receptor produces a conformational change in the insulin receptor which increases insulin receptor affinity, insulin sensitivity and acts on an early post-receptor event responsible for coupling binding to insulin action.