Small quantities of ionic surfactants modify the crystallization of lysozyme and lead to the formation of phases with different morphologies. By using fluorescent probes, we demonstrate that the surfactants are incorporated into the growing crystals (which is a possible reason for crystal twinning) and can penetrate and adsorb within previously formed crystals. To characterize the interactions and the mass transfer of surfactants inside the crystalline protein matrices, we studied the infusion of lysozyme crystals with pyrene-based fluorescent surfactants by quantitative fluorescence microscopy. The fluorescence intensity data were fitted to the diffusion equation to obtain the effective diffusion coefficient of the fluorophore in the crystals. The diffusion coefficients obtained range from 2 × 10-10 to 30 × 10-10 cm2/s, depending on the type and size of the surfactant. The slow infusion is a consequence of the strong surfactant adsorption on the protein lattice; the estimated energy of adsorption is 10-12 kT. Fluorescence recovery after photobleaching (FRAP) experiments with saturated crystals showed that surfactant self-diffusion in these crystals is negligible, which is yet another indication of strong adsorption. The data shed light on the interactions between condensed protein phases and surfactants and are relevant to protein purification and crystallization and to the manipulation of proteins by light.