The relationship between local mass transfer coefficient and fluid velocity in heterogenous biofilms was investigated by combining microelectrodes and confocal scanning laser microscopy (CSLM). The biofilms were grown for up to 7 days and consisted of cell clusters separated by interstitial channels. Mass transfer coefficient depth profiles were measured at specific locations in the cell clusters and channels at average flow velocities of 2.3 and 4.0 cm/s. Liquid flow velocity profiles were measured in the same locations using a particle tracking technique. The velocity profiles showed that flow in the open channel was laminar. There was no flow at the top surface of the biofilm cell clusters but the mass transfer coefficient was 0.01 cm/s. At the same depth in a biofilm channel, the flow velocity was 0.3 cm/s and the mass transfer coefficient was 0.017 cm/s. The mass transfer coefficient profiles in the channels were not influenced by the surrounding cell clusters. Local flow velocities were correlated with local mass transfer coefficients using a semi-theoretical mass transfer equation. The relationship between the Sherwood number (Sh,) the Reynolds number (Re,) and the Schmidt number (Sc) was found using the experimental data to find the dimensionless empirical constants (n1, n2, and m) in the equation Sh = n(1) + n(2) Re-m Sc-1/3 The values of the constants ranged from 1.45 to 2.0 for n(1), 0.22 to 0.28 for n(2), and 0.21 to 0.60 for m. These values were similar to literature values for mass transfer in porous media. The Sherwood number for the entire flow cell was 10 when the bulk flow velocity was 2.3 cm/s and 11 when the bulk flow velocity was 4.0 cm/s.