Release of Ca(2+) with dipicolinic acid (CaDPA) was monitored by Raman spectroscopy and differential interference contrast microscopy during germination of individual spores of Bacillus subtilis strains with alterations in GerD and SpoVA proteins. Notable conclusions about germination after the addition of nutrient were as follows. (i) Following L-alanine addition, wild-type and gerD spores and spores with elevated SpoVA protein levels (↑SpoVA spores) slowly released ∼10% of their CaDPA during a variable (6- to 55-min) period ending at T(lag), the time when faster CaDPA release began. (ii) T(lag) times were lower for ↑SpoVA spores than for wild-type spores and were higher for gerD spores. (iii) The long T(lag) times of gerD spores were partially due to slow commitment to germinate. (iv) The intervals between the commitment to germinate and CaDPA release were similar for wild-type and ↑SpoVA spores but longer for gerD spores. (v) The times for rapid CaDPA release, ΔT(release) = T(release) - T(lag) (with T(release) being the time at which CaDPA release was complete), were similar for wild-type, gerD, and ↑SpoVA spores. (vi) Spores with either one of two point mutations in the spoVA operon (spoVA(1) and spoVA(2) spores) exhibited a more rapid rate of CaDPA release beginning immediately after L-alanine addition leading to ∼65% CaDPA release prior to T(lag). (vii) T(lag) times for spoVA(1) and spoVA(2) spores were longer than for wild-type spores. (viii) The intervals between spoVA(1) and spoVA(2) spores' commitment and CaDPA release were similar to those for wild-type spores, but commitment occurred later. In contrast to germination after the addition of nutrient, T(lag) and ΔT(release) times were relatively similar during dodecylamine germination of spores of the five strains. These findings suggest the following. (i) GerD plays no role in CaDPA release during spore germination. (ii) SpoVA proteins are involved in CaDPA release during germination with nutrients, and probably with dodecylamine. (iii) Spores release significant CaDPA before commitment. (iv) CaDPA release during T(lag) and ΔT(release) may signal subsequent germination events.