We have used X-ray microangiography to investigate the hypothesis that the potent endogenous vasodilator endothelium-derived relaxing factor (EDRF) contributes to the maintenance of "optimality" in vascular branching by modulating the diameters of the parent (D0) and daughter (D1 and D2) arteries at bifurcations. Five anatomically different types of bifurcation were studied in buffer-perfused rabbit ear preparations both under resting conditions and after pharmacological constriction by 5-hydroxytryptamine (5HT). A range of flow rates (1-5 ml min-1) was employed as release of EDRF from endothelial cells is stimulated by shear stress. Experimental data obtained in the presence and absence of EDRF activity were compared with theoretical predictions in three ways. (1) Junction exponents (x) were determined at each bifurcation from the equation Dx1 + Dx2 = Dx0, and their frequency distributions constructed. Murray (1926a, Proc. natn. Acad. Sci., U.S.A. 12, 207-214; 1926b, J. gen. Physiol. 9, 835-841.) proposed that x will be exactly 3 if power losses and intravascular volume are minimized simultaneously. In unconstricted preparations, either in the presence or absence of EDRF activity, and in preparations constricted by 0.1 microM 5HT in the presence of EDRF activity, the modes and medians of the frequency distributions of x were found to be close to 3 at all flow rates. In contrast, in 0.1 microM 5HT-constricted preparations in the absence of EDRF activity, no single mode common to all flow rates was apparent and medians were significantly larger at all flow rates. (2) Theoretically "optimal" branching angles were derived from experimental diameter measurements using four mathematical models which minimize respectively the total surface area, total volume, total drag (shear stress) and total power losses at bifurcations (Murray, 1926b). These calculated branching angles were then compared with actual branching angles. EDRF activity was found to be necessary for accurate prediction of branching angles by the minimum volume and power loss models in 5HT-constricted but not in resting preparations. (3) For each model or "minimization principle", there is an optimal mathematical relationship between the junction exponent, x, and the angle between daughter arteries, psi 12, at a bifurcation (Roy & Woldenberg, 1982, Bull. math. Biol. 44, 349-360.) Experimentally determined values of x and psi 12 agreed closely with those predicted both by the minimum volume and the minimum power loss principles, except again in 5HT-constricted preparations in the absence of EDRF activity.(ABSTRACT TRUNCATED AT 250 WORDS)