The most important group of antifungals is the azoles (e.g. miconazole), which act by inhibiting lanosterol demethylase in the sterol biosynthesis pathway. Azole activity can be modulated through structural changes in lanosterol demethylase, altered expression of its gene ERG11, alterations in other sterol biosynthesis enzymes or altered expression of multidrug transporters. We present evidence that azole activity versus Saccharomyces cerevisiae is also modulated by Ca2+-regulated signalling. (i) Azole activity was reduced by the addition of Ca2+. Conversely, azole activity was enhanced by the addition of Ca2+ chelator EGTA. (ii) Three structurally distinct inhibitors (fluphenazine, calmidazolium and a W-7 analogue) of the Ca2+-binding regulatory protein calmodulin enhanced azole activity. (iii) Two structurally distinct inhibitors (cyclosporin and FK506) of the Ca2+-calmodulin-regulated phosphatase calcineurin enhanced azole activity. (iv) Strains in which the Ca2+ binding sites of calmodulin were eliminated and strains in which the calcineurin subunit genes were disrupted demonstrated enhanced azole sensitivity; conversely, a mutant with constitutively activated calcineurin phosphatase demonstrated decreased azole sensitivity. (v) CRZ1/TCN1 encodes a transcription factor regulated by calcineurin phosphatase; its disruption enhanced azole sensitivity, whereas its overexpression decreased azole sensitivity. All the above treatments had comparable effects on the activity of terbinafine, an inhibitor of squalene epoxidase within the sterol biosynthesis pathway, but had little or no effect on the activity of drugs with unrelated targets. (vi) Treatment of S. cerevisiae with azole or terbinafine resulted in transcriptional upregulation of genes FKS2 and PMR1 known to be Ca2+ regulated. A model to explain the role of Ca2+-regulated signalling in azole/terbinafine tolerance is proposed.