Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca(2+)-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of approximately 20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior.