A rapid diagnosis of a biological threat in a powder sample is important for ﬁrst responders who have to make decisions on-site. The present culture-based method does not provide timely results, which is a critical barrier for a quick response when a suspicious powder sample is found. The ATP bioluminescence method, combined with a heat shock, was investigated to determine the presence of spores in powder. The results show that only spore-containing powder samples provided a dramatic increase in the bioluminescence signal after the heat shock, which induces germination of the spores. Various conditions were tested to ﬁnd the most effective and rapid germination procedure. Elevated temperatures (37°C and 50°C) were more effective in germination than room temperature. At 50°C, a double-strength germinant was more effective in germination than the regular strength. The 37°C/15 min procedure induced the germination of spores most effectively, while a 50°C/2 min procedure provided reasonably high signals, so it could make the entire procedure even faster (< 5 min). The detection limit of the bioluminescence method is < 100 spores. Copyright © 2004 John Wiley & Sons, Ltd.