Heparan sulfate proteoglycans (HSPGs) are concentrated at neuromuscular synapses in many species, including Drosophila. We have established the physiological and patterning functions of HSPGs at the Drosophila neuromuscular junction by using mutations that block heparan sulfate synthesis or sulfation to compromise HSPG function. The mutant animals showed defects in synaptic physiology and morphology suggesting that HSPGs function both presynaptically and postsynaptically; these defects could be rescued by appropriate transgene expression. Of particular interest were selective disruptions of mitochondrial localization, abnormal distributions of Golgi and endoplasmic reticulum markers in the muscle, and a markedly increased level of stimulus-dependent endocytosis in the motoneuron. Our data support the emerging view that HSPG functions are not limited to the cell surface and matrix environments, but also affect a diverse set of cellular processes including membrane trafficking and organelle distributions.