Analysis of upwelling radiation (spectral reflectance) by remote sensing may provide valuable information on the nature and distribution of the primary producers, the microphytobenthos, on intertidal mudflat systems. Spatial and temporal variation in the spectral reflectance signal (400-900 nm) from the surface of an exposed intertidal mudflat was investigated in relation to the density and vertical position of microphytobenthos assemblages within the sediment. Spatial measurements were obtained from areas of sediment colonized by different assemblages of microphytobenthos (mainly mixtures of diatoms and euglenids). In addition, a station was selected to examine temporal changes in spectral reflectance (corrected for variations in ambient light) over an exposure period. It was shown that the spectral reflectance signal varied, depending on the pigment compliment and biomass of the surface assemblages of microphytobenthos. The qualitative variation in composition of the assemblages visualized by low-temperature scanning electron microscopy was confirmed by analysis of species composition (light microscopy) and by pigment fingerprinting (diode array high-performance liquid chromatography [HPLC]). Time-series analysis showed that the migration of cells to the surface of the sediment during a daytime emersion period rapidly changed the optical properties of the sediment surface. Analysis of sediment pigment content was conducted using standard surface scrapes (0.5-cm depth) and a high resolution sectioning technique (200-μm layers). The migration influence was only detected by fine-scale analysis of pigments. This was demonstrated by a step-wise elimination analysis, which showed that correlations between the absorbance characteristics of the sediment and pigment content improved as premigration data were excluded. Maximum light penetration into the natural sediment was of the order of 2 mm under light conditions comparable with ambient levels. Two main conclusions were drawn from this study: (1) interpretation of spectral reflectance data must be consistent with knowledge of the ecology and behavior (cyclic migration patterns) of intertidal microphytobenthos, and (2) ground truthing of pigment-related signals from intertidal flats should be conducted on a scale relevant to the process of spectral reflectance from sediments. The implications of these findings are discussed.