Frequenin (Frq) and its mammalian homologue, neuronal calcium sensor 1 (NCS-1), are important calcium-binding proteins which enhance neurotransmitter release and facilitation. Here, we report the discovery of a second Frq-encoding gene (frq2) in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Frq1 is more abundant than Frq2, and is most highly expressed in larva. Loss-of-function phenotypes were studied using dominant negative peptides to prevent Frq target binding, RNAi to reduce gene transcription, or both methods. To discriminate chronic from acute loss-of-function effects, we compared the effects of transgenic expression and forward-filling the dominant-negative peptide into presynaptic terminals. In both cases, a 70% reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations of the synapse during development. The chronic treatment also produced more synaptic boutons from MNSNb/d-Is motorneurons, but fewer active zones per bouton. By contrast, excess-of-function conditions yielded a 1.4- to 2-fold increase in quantal content and fewer boutons in the same motorneuron. These synaptic effects resulted in behavioural changes in the Buridan locomotion assay, showing that walking speed is dependent on Frq activity in the nervous system. All the effects were identical for both Frqs, and consistent with excess- and loss-of-function genotypes. We conclude that Frqs have two distinct functions: one in neurotransmission, regulating the probability of release per synapse, and another in axonal growth and bouton formation.