During metamorphic development, the arrival at the olfactory (antennal) lobe of olfactory receptor axons initiates the process of glomerulus formation. The glomeruli are discrete spheroidal regions of neuropil that are the sites of synaptic interactions among receptor neurons and their target antennal-lobe neurons. The process of glomerulus formation begins as groups of receptor axons form protoglomeruli. These dense clusters of terminal branches mostly are discrete entities from the time they can be recognized, although a few branches from neighboring protoglomeruli overlap laterally. A previous study by Schweitzer et al. [Schweitzer E. S., Sanes J. R. and Hildebrand J. G. (1976) Ontogeny of electroantennogram responses in the moth, Manduca sexta. J. Insect Physiol. 22, 955-960] has shown that odor-induced activity in the receptor neurons can be detected first in recordings from the axons in the antennal nerve only in the last few days of metamorphic development and thus could not influence the process of glomerulus formation. In this study, we have tested directly the possibility that an earlier presence of spontaneous activity in either the receptor axons or the antennal-lobe neurons could affect the process. Tetrodotoxin, a Na(+)-channel blocker, was injected into the hemolymph prior to the onset of glomerulus formation to block any spontaneous Na(+)-dependent activity. Subsequent intracellular recordings from antennal-lobe neurons revealed no spike activity. Comparison with vehicle-injected control animals at stages during and after glomerulus formation revealed no differences in the localization of receptor-axon terminal branches in the glomeruli, in the border of glial cells that forms around each glomerulus, or in the morphology of the tufted glomerular arbors of one of the antennal-lobe neurons. We conclude that: (1) the process of glomerulus formation is largely independent of activity; and (2) glomeruli as modular units of the CNS more closely resemble cortical barrels than cortical columns, both in their ontogeny and in the lack of an obvious effect of activity on the morphology of the neurons arborizing within them.