The present study represents the first broader evaluation of the rapid 30-s kinetic bioluminescence assay with Vibrio fisheri (microplate format modification) for contact toxicity testing of whole sediments. The present study focused on river sediments from the Morava River basin, Czech Republic, repeatedly sampled during 2005 to 2006 and analyzed for geological and geochemical parameters, content of toxic metals, major organic pollutants, and toxicity. High natural variation in toxicity (50% inhibitory concentration [IC50] values ranging from 0.8 to >80 mg sediment dry wt/ml) was found (among different sampling periods and years, among sites), and this could be related to the sediment dynamics affected by spring high flows and summer droughts. From the 46 sediment descriptors, exchangeable protons (H(+)) was the only parameter that consistently correlated with toxicity. Three other descriptors (i.e., content of organic carbon plus two parameters from the detailed silicate analysis of sediments: percentage of SO(3) representing total sulfur content, structural water H(2)O+) also significantly correlated with toxicity. There were only minor and variable correlations with contamination. We propose sediment safety guideline categories for the V. fisheri kinetic test with severe toxicity threshold of IC50 < 1 mg dry wt/ml. Although sediments are considered a rather stable matrix in comparison with river water, we confirmed high variability and dynamics that should be reflected in monitoring plans and field studies.