Oxygen limitation in solid-state fermentation (SSF) has been the topic of modeling studies, but thus far, there has been no experimental elucidation on oxygen-transfer limitation at the particle level. Therefore, intra-particle oxygen transfer was experimentally studied in cultures of Rhizopus oligosporus grown on the surface of solid, nutritionally defined, glucose and starch media. The fungal mat consisted of two layers-an upper layer with sparse aerial hyphae and gas-filled interstitial pores, and a dense bottom layer with liquid-filled pores. During the course of cultivation ethanol was detected in the medium indicating that oxygen was depleted in part of the fungal mat. Direct measurement of the oxygen concentrations in the fungal mat during cultivation, using oxygen microelectrodes, showed no oxygen depletion in the upper aerial layer, but revealed development of steep oxygen concentration gradients in the wet bottom layer. Initially, the fungal mat was fully oxygenated, but after 36.5 hours oxygen was undetectable at 100 mum below the gas-liquid interface. This was consistent with the calculated oxygen penetration depth using a reaction-diffusion model. Comparison of the overall oxygen consumption rate from the gas phase to the oxygen flux at the gas-liquid interface showed that oxygen consumption of the microorganisms occurred mainly in the wet part of the fungal mat. The contribution of the aerial hyphae to overall oxygen consumption was negligible. It can be concluded that optimal oxygen transfer in SSF depends on the available interfacial gas-liquid surface area and the thickness of the wet fungal layer. It is suggested that the moisture content of the matrix affects both parameters and, therefore, plays an important role in optimizing oxygen transfer in SSF cultures.