We have analyzed the intracellular and cell-to-cell diffusion kinetics of fluorescent tracers in the Chironomus salivary gland. We use this analysis to investigate whether membrane potential-induced changes in junctional permeability are accompanied by changes in cell-to-cell channel selectivity. Tracers of different size and fluorescence wavelength were coinjected into a cell, and the fluorescence was monitored in this cell and an adjacent one. Rate constants, kj, for cell-to-cell diffusion were derived by compartment model analysis, taking into account (i) cell-to-cell diffusion of the tracers; (ii) their loss from the cells; (iii) their binding (sequestration) to cytoplasmic components; and (iv) their relative mobility to cytoplasm, as determined separately on isolated cells. In cell pairs, we compared a tracer's kj with the electrical cell-to-cell conductance, gj. At cell membrane resting potential, the kj's ranged 3.8-9.2 X 10(-3) sec-1 for the small carboxyfluorescein (mol wt 376) to about 0.4 X 10(-3) sec-1 for a large fluorescein-labeled sugar (mol wt 2327). Cell membrane depolarization reversibly reduced gj and kj for a large and a small tracer, all in the same proportion. This suggests that membrane potential controls the number of open channels, rather than their effective pore diameter or selectivity. From the inverse relation between tracer mean diameter and relative kj we calculate an effective, permeation-limiting diameter of approximately 29 A for the insect cell-to-cell channel. Intracellular diffusion was faster than cell-to-cell diffusion, and it was not solely dependent on tracer size. Rate constants for intracellular sequestration and loss through nonjunctional membrane were large enough to become rate-limiting for cell-to-cell tracer diffusion at low junctional permeabilities.