Chloroquine inhibited the outgrowth of Bacillus subtilis spores at a 10-fold lower concentration than that required to prevent vegetative growth. Analysis of macromolecular synthesis in outgrowing spores and vegetative cells in the presence of chloroquine indicated that it acted preferentially on transcription. Differential sensitivity of outgrowing spores and vegetative cells to chloroquine was not due to changes in the specificity of the RNA-polymerase, since RNA-polymerase activity measured in permeabilized cells was not affected differently by the drug. The preferential inhibition of spore outgrowth was not evident at pH 8.0, at which the majority of chloroquine is in a monovalent, more lipophilic, form. In the presence ofinhibitors affecting membrane potential, vegetative cells were as sensitive to chloroquine as outgrowing spores. Measurement of [14C]chloroquine uptake showed that early outgrowing spores accumulated twice as much drug as resistant late outgrowing spores and seven times more than vegetative cells. Treatment of vegetative cells with metabolic inhibitors led them to accumulate chloroquine to the levels found in outgrowing spores. Therefore, the preferential inhibition of outgrowing spores by chloroquine is the result of increased uptake of the drug, reflecting differences in energy metabolism from vegetative cells.