The nitrogen-fixing actinobacterium Frankia develops unique multicellular structures called vesicles, which are the site of nitrogen fixation. These vesicles are surrounded by a thick hopanoid lipid envelope that protects nitrogenase against oxygen inactivation. The phenotypes of five mutants that form smaller numbers of vesicles were investigated. The vesicles of these mutants were smaller than those of the wild type and had a phase dark appearance. They induced the expression of a glutamine synthetase gene in hyphae cells in response to ammonium starvation. These results suggest that genes impaired in the mutants do not function in global nitrogen regulation, but specifically function in vesicle differentiation.