Lactate is produced in most vertebrate cells as a by-product of anaerobic metabolism. In addition to its role as a fuel for many tissues, circulating lactate can act as a signalling molecule and stimulates ventilation in air- and water-breathing vertebrates. Recent evidence suggests lactate acts on O2- and CO2/H+-sensitive chemoreceptors located in the mammalian carotid body. While analogous receptors (neuroepithelial cells or NECs) in fish gills are presumed to also function as lactate sensors, direct evidence is lacking. Here, using ratiometric Fura-2 Ca2+ imaging, we show that chemosensitive NECs isolated from killifish gills respond to lactate (5–10 mmol l−1; pHe ∼7.8) with intracellular Ca2+ elevations. These responses were inhibited by an L-type Ca2+ channel blocker (nifedipine; 0.5 µmol l−1), a monocarboxylic acid transporter (MCT) blocker (α-cyano-4-hydroxycinnamate; 300 µmol l−1) or a competitive MCT substrate (pyruvate; 5 mmol l−1). These data provide the first direct evidence that gill NECs act as lactate sensors.