Levinson Hyatt (1966) showed that the measurable events collectively recognized as spore germination, and previously thought to take place concurrently, occurred in Bacillus megaterium QM BI~~I in the following sequence: loss of resistance to heat and mercuric chloride; release of dipicolinic acid (DPA) ; onset of stainability; darkening of individual spores under phase contrast optics and fall in extinction of spore suspen- sions. Although the mechanism initiating these changes is not known, Strange & Dark (1957) and Gould, Hitchins & King (1966) suggested that they result more or less directly from depolymerization of peptidoglycan in the spore cortex, catalysed by a lytic enzyme which can be extracted from spores of some species and which can be shown to cause germination-like changes in chemically sensitized spores (Gould & King, 1969). If this is so then the excretion of peptidoglycan fragments would be expected to be one of the earliest determinable events during germination. However, if hydrolysis of peptidoglycan only commences following germination, then excretion of fragments should occur late in the sequence of events. The experiments described here were designed to resolve these possibilities. Organism. Spores of Bacillus cereus T were grown in G medium (Stewart & Halvor- son, I 953). Peptidoglycan in spores was labelled with generally tritiated a,6-diamino- pimelic acid (rH) DAP ; Radiochemical Centre, Amersham, Buckinghamshire) by adding (3H) DAP (40 pm. ; specific activity, 9 mCi/mmole) and cold lysine (I mM) to cultures in the 'granular' stage of growth, just prior to the commencement of sporu- lation. The presence of excess lysine prevented decarboxylation of the (3H)DAP (Vinter, 1965). Spores were cleaned by centrifuging six times in ice-cold HCI (0.03~) as recommended by Murrell & Warth (1969, twice in ice-cold distilled water, and stored frozen. Germination. Spores were activated by heating in water at 70' for 30 min., washed once in water, then added (about Io8/ml.) to the following solution at 30°, to initiate germination: tris (hydroxymethyl amino methane) + HCl buffer (100 mM, pH 8.0), containing L-alanine (5 mM) and inosine (5 mM). Samples were taken at intervals, and the germination process was rapidly halted either by separating spores from the supernatant fraction in about 15 sec. by membrane filtration using filter membranes (0.45 pm. mean pore size) in Swinney filter holders on syringe barrels (Millipore Corp., Bedford, Massachusetts, U.S.A.), or by adding formaldehyde (20 % v/v) and quickly centrifuging. AnaZyses. Pellet and supernatant samples were analysed for calcium using an Atomic Absorption Spectrophotometer (Unicam Instruments Ltd, Cambridge). Dipicolinic