Summary: The ability of 13 amino-group-modified and 12 carboxyl-group-modified L-alanine derivatives to initiate germination of Bacillus subtilis spores was examined. The ‘relative affinity’, defined as the ratio of the concentration of L-alanine required to give 20% of the maximal germination rate to the concentration of analogue required to give this same rate, was used to estimate the effectiveness of the analogues as germinants. L-Ala-L-Ala had a relative affinity of only 1/4200. For most of the L-alanyl dipeptides studied (with the exception of L-Ala-L-Pro and L-Pro-L-Ala), their effectiveness as germinants was lower if the second amino acid was substituted in the carboxyl group rather than the amino group of L-alanine. N-(2-Methylsulphonyl)ethyloxy-carbonyl-L-Ala was a good germinant (relative affinity 1/100), indicating that there may be an additional complementary site for this group in the germinant receptor field on the spore. Compounds with a shorter chain, N-t-butoxycarbonyl-L-Ala and N-acetyl-L-Ala, showed lower affinities, and compounds with an aromatic ring were very poor germinants. Replacement of the carboxyl group of L-alanine by an alcohol group caused a total loss of germinant activity, and its modification by conversion to an amide group or by substitution of amino acids resulted in low affinity and germination rates. Esterified L-alanine derivatives retained high activity, suggesting that retaining an electronegative group was important. The stimulatory effect of glucose on germination was very marked for compounds with a hydrophobic substituent in either the amino or the carboxyl group, such as L-Leu-L-Ala, L-Ala-L-Leu, N-carbobenzoxy-L-Ala and N-dinitrophenyl-L-Ala. Overall, it can be deduced that both -NH - and -COO- groups, separated by 1 or 2 interatomic distances, are important for germinant activity. Substituents in the amino group were tolerated better than those in the carboxyl group.