Picrotoxin is extensively and specifically used to inhibit GABAA receptors and other members of the Cys-loop receptor superfamily. We find that picrotoxin acts independently of known Cys-loop receptors to shorten the period of the circadian clock markedly by specifically advancing the accumulation of PERIOD2 protein. We show that this mechanism is surprisingly tetrodotoxin-insensitive, and the effect is larger than any known chemical or genetic manipulation. Notably, our results indicate that the circadian target of picrotoxin is common to a variety of human and rodent cell types but not Drosophila, thereby ruling out all conserved Cys-loop receptors and known regulators of mammalian PERIOD protein stability. Given that the circadian clock modulates significant aspects of cell physiology including synaptic plasticity, these results have immediate and broad experimental implications. Furthermore, our data point to the existence of an important and novel target within the mammalian circadian timing system. © 2013 the American Physiological Society.