Acetaminophen (paracetamol), one of the most widely used analgesics, is toxic under conditions of overdose or in certain disease conditions, but the mechanism of acetaminophen toxicity is still not entirely understood. To obtain fresh insights into acetaminophen toxicity, this phenomenon was investigated in yeast. Acetaminophen was found to be toxic to yeast cells, withergmutants displaying hypersensitivity. Yeast cells grown in the presence of acetaminophen were found to accumulate intracellular acetaminophen, but no metabolic products of acetaminophen could be detected in these extracts. The toxicity response did not lead to an oxidative stress response, although it did involve Yap1p. The cytochrome P450 enzymes of yeast, Erg5p and Erg11p, did not appear to participate in this process, unlike the mammalian systems. Furthermore, we could not establish a central role for glutathione depletion or the cellular glutathione redox status in acetaminophen toxicity, suggesting differences from mammalian systems in the pathways causing toxicity. Investigations of the resistance mechanisms revealed that deletion of the glutathione-conjugate pumps Ycf1p (a target of Yap1p) and Bpt1p, surprisingly, led to acetaminophen resistance, while overexpression of the multidrug resistance pumps Snq2p and Flr1p (also targets of Yap1p) led to acetaminophen resistance. The Yap1p-dependent resistance to acetaminophen required a functional Pdr1p or Pdr3p protein, but not a functional Yrr1p. In contrast, resistance mediated by Pdr1p/Pdr3p did not require a functional Yap1p, and revealed a distinct hierarchy in the resistance to acetaminophen.