Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca(2+)-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca(2+) entry and enhanced nerve-terminal growth. The impaired Ca(2+) entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca(2+) channels, or that it regulates the PI4Kbeta pathway as described in other organisms. To determine whether Frq interacts with PI4Kbeta with consequent effects on Ca(2+) channels, we first characterized a PI4Kbeta-null mutant and found that PI4Kbeta was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4Kbeta-null background. We conclude that the effects of Frq are not due to an interaction with PI4Kbeta. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the alpha(1)-subunit of voltage-gated Ca(2+) channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca(2+) entry through a functional interaction with the alpha(1) voltage-gated Ca(2+)-channel subunit; this interaction regulates neurotransmission and nerve-terminal growth.