Abstract— We have reinvestigated the role of aldehyde in bacterial bioluminescence relative to its absolute requirement for light emission. We have found that aldehyde is an absolute requirement for light emission at 25°C as well as in the frozen state (—3° to — 9°C). As found by earlier workers, certain luciferase preparations isolated from Ph. Jischeri do not appear to require aldehyde for bioluminescence from the frozen state. We can now attribute this behavior to contaminating levels of aldehyde in those preparations. The results suggest that reactant puddling exist in the frozen state in which micro regions of liquid form within the ice crystals resulting in enormous increases in reactant concentration. Copyright © 1969, Wiley Blackwell. All rights reserved