Synopsis The mechanoreceptive lateral line system in fish is composed of neuromasts containing hair cells, which can be temporarily ablated by aminoglycoside antibiotics and heavy metal ions. These chemicals have been used for some time in studies exploring the functional role of the lateral line system in many fish species. However, little information on the relative effectiveness and rate of action of these chemicals for ablation is available. In particular, aminoglycoside antibiotics are thought to affect canal neuromasts, which sit in bony or trunk canals, differently from superficial neuromasts, which sit directly on the skin. This assumed ablation pattern has not been fully quantified for commonly used lateral line ablation agents. This study provides a detailed characterization of the effects of two aminoglycoside antibiotics, streptomycin sulfate and neomycin sulfate, and a heavy metal salt, cobalt (II) chloride hexahydrate (CoCl2), on the ablation of hair cells in canal and superficial neuromasts in the giant danio (Devario aequipinnatus) lateral line system, as a model for adult teleost fishes. We also quantified the regeneration of hair cells after ablation using CoCl2 and gentamycin sulfate to verify the time course to full recovery, and whether the ablation method affects the recovery time. Using a fluorescence stain, 4-Di-2-ASP, we verified the effectiveness of each chemical by counting the number of fluorescing canal and superficial neuromasts present throughout the time course of ablation and regeneration of hair cells. We found that streptomycin and neomycin were comparably effective at ablating all neuromasts in less than 12 h using a 250 μM dosage and in less than 8 h using a 500 μM dosage. The 500 μM dosage of either streptomycin or neomycin can ablate hair cells in superficial neuromasts within 2–4 h, while leaving those in canal neuromasts mostly intact. CoCl2 (0.1 mM) worked the fastest, ablating all of the hair cells in less than 6 h. Complete regeneration of the neuromasts in the lateral line system took 7 days regardless of chemicals used to ablate the hair cells. This study adds to the growing knowledge in hearing research about how effective specific chemicals are at ablating hair cells in the acoustic system of vertebrates.