Synapse formation requires the gradual accumulation of cytomatrix proteins and voltage-gated Ca2+ channels (VDCCs) at presynaptic active zones (AZs) to support neurotransmitter release. To correlate AZ maturation with synaptic output, quantal imaging was performed at serially imaged time-stamped Drosophila synapses. Evoked release strength correlated strongly with AZ age and accumulation of late AZ scaffolds, while immature sites lacking VDCC accumulation supported spontaneous release. To examine how neuronal activity regulates AZ maturation and protein accumulation, the effects of disruptions to SV fusion or action potential generation were analyzed. Decreasing neuronal activity reduced AZ seeding and caused hyperaccumulation of presynaptic material at existing AZs. Although enlarged AZs are also observed in rab3 mutants, activity reduction acted through an independent mechanism that required postsynaptic glutamate receptor-dependent signaling. Together, these data indicate AZ maturation state sets distinct presynaptic release modes and output strength, with neuronal activity shaping both AZ number and size across development. ### Competing Interest Statement J.M. is an employee of Abberior Instruments America, which commercializes super-resolution microscopy systems, including MINFLUX.