Two questions bearing on the use of fura-2 to measure transient changes in intracellular Ca2+ concentration have been addressed. To investigate fura-2 intracellular binding, the amounts of fura-2 and [14C]glycine in Balanus nubilus myofibrillar bundles after loading were determined and their intracellular apparent diffusion constants measured. No significant fura-2 immobilisation occurs under the conditions used. The apparent diffusion constant for fura-2 in aqueous solution was determined. The relationship between half-time for relaxation of force and fura-2 fluorescence transients, and intracellular fura-2 concentration, in voltage-clamped single muscle fibres was examined. Significant buffering of the Ca2+ transient occurred at fura-2 concentrations above approximately 6 microM.