Clusters of calcium-loaded sarcoplasmic reticulum (SR) vesicles in agarose gel were previously shown to behave as an excitable medium that propagates calcium waves. In a 3D-hexagonal disposition, the distance between neighboring spheres (which may stand for SR vesicles) is constant and the relationship between distance and vesicular protein concentration is expected to be nonlinear. To obtain a distribution of SR vesicles at different protein concentrations as homogeneous as possible, liquid agarose gels were carefully stirred. Electron micrographs, however, did not confirm the expected relationship between inter-SR vesicle distance and vesicular protein concentration. Light micrographs, to the contrary, resulted in a protein concentration-dependent disposition of clusters of SR vesicles, which is described by a linear function. Stable calcium waves in agarose gel occurred at SR vesicle protein concentrations between 7 and 16 g/l. At lower protein concentrations, local calcium oscillations or abortive waves were observed. The velocities of calcium waves were optimum at approximately 12 g/l and amounted to nearly 60 microm/s. The corresponding distance of neighboring calcium release units was calculated to be approximately 4 microm. The results further show that calcium signaling in the described reaction-diffusion system is optimal in a relatively small range of diffusion lengths. A change by +/-2 microm resulted in a reduction of the propagation velocity by 40%. It would appear that 1), the distance between calcium release units (clusters of ryanodine receptors in cells) is a sensitive parameter concerning propagation of Ca2+ signals; and 2), a dysfunction of the reaction-diffusion system in living cells, however, might have a negative effect on the spreading of intracellular calcium signals, thus on the cell's function.