Mode of action studies were made with Hoe 296, a new synthetic antimycotic, mainly in Candida albicans. The specific parameters examined included possible effects on (i) osmotic fragility, (ii) respiration, (iii) uptake and incorporation of radioactive leucine and adenine, and (iv) leakage or efflux of intracellular K+ and other materials. As a result, fungitoxic activity of Hoe 296 appears to be attributed to inhibition of uptake of precursors of macromolecular syntheses from the medium. Uptake and accumulation of leucine in the internal pool of starved cells was more susceptible to the drug than subsequent incorporation into proteins. Hoe 296 was also effective in altering the cell permeability, but greater drug concentrations were required to induce appreciable leakage of cellular constituents, such as 260 nm-absorbing materials, folin-positive substances and potassium ions, from the cells. Osmotic fragility or endogenous respiration was virtually insensitive to the drug. Partial inhibition by relatively high concentrations of Hoe 296 of the respiratory activity of yeast cells or mitochondria therefrom with exogenous substrates can be explained by decreased uptake of the substrates from the medium.