Aims: Limited information is available on the germination triggers for spores of nonproteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. Methods and Results: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-Cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40degreesC, but not at 50degreesC, and was optimal at 20-25degreesC. Conclusions: The automated system enabled a systematic study of germination requirements. and provided an insight into germination in spores of non-proteolytic Cl. botulinum. Significance and Impact of the Study: The results extend understanding of germination of C, non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.