Volatile anesthetics including isoflurane affect all cells examined, but their mechanisms of action remain unknown. To investigate the cellular basis of anesthetic action, we are studying Saccharomyces cerevisiae mutants altered in their response to anesthetics. The zzz3-1 mutation renders yeast isoflurane resistant and is an allele of GCN3. Gcn3p functions in the evolutionarily conserved general amino acid control (GCN) pathway that regulates protein synthesis and gene expression in response to nutrient availability through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). Hyperphosphorylation of eIF2alpha inhibits translation initiation during amino acid starvation. Isoflurane rapidly (in <15 min) inhibits yeast cell division and amino acid uptake. Unexpectedly, phosphorylation of eIF2alpha decreased dramatically upon initial exposure although hyperphosphorylation occurred later. Translation initiation was inhibited by isoflurane even when eIF2alpha phosphorylation decreased and this inhibition was GCN-independent. Maintenance of inhibition required GCN-dependent hyperphosphorylation of eIF2alpha. Thus, two nutrient-sensitive stages displaying unique features promote isoflurane-induced inhibition of translation initiation. The rapid phase is GCN-independent and apparently has not been recognized previously. The maintenance phase is GCN-dependent and requires inhibition of general translation imparted by enhanced eIF2alpha phosphorylation. Surprisingly, as shown here, the transcription activator Gcn4p does not affect anesthetic response.