Choline- O -sulfate uptake by Penicillium notatum showed the following characteristics. (i) Transport was mediated by a permease which is highly specific for choline- O -sulfate. No significant inhibition of transport was caused by choline, choline- O -phosphate, acetylcholine, ethanolamine- O -phosphate, ethanolamine- O -sulfate, methanesulfonyl choline, 2-aminoethane thiosulfate, or the monomethyl or dimethyl analogues of choline- O -sulfate. Similarly, no significant inhibition was caused by any common sulfur amino acid or inorganic sulfur compound. Mutants lacking the inorganic sulfate permease possessed the choline- O -sulfate permease at wild-type levels. (ii) Choline- O -sulfate transport obeyed saturation kinetics ( Km = 10 −4 to 3 × 10 −4 m ; Vmax = 1 to 6 μmoles per g per min). The kinetics of transport between 10 −9 and 10 −1 m external choline- O -sulfate showed that only one saturable mechanism is present. (iii) Transport was sensitive to 2,4-dinitrophenol, azide, N -ethylmaleimide, p -chloromercuribenzoate, and cyanide. Ouabain, phloridzin, and eserine had no effect. (iv) Transport was p H-dependent with an optimum at p H 6. Variations in the ionic strength of the incubation medium had no effect. (v) Transport was temperature-dependent with a Q 10 of greater than 2 between 3 and 40 C. Transport decreased rapidly above 40 C. (vi) Ethylenediaminetetraacetate (sodium salts, p H 6) had no effect, nor was there any stimulation by metal or nonmetal ions. Cu ++ , Ag + , and Hg ++ were inhibitory. (vii) The initial rate at which the ester is transported was independent of intracellular hydrolysis. After long periods of incubation (> 10 min), a significant proportion of the transported choline- O -sulfate was hydrolyzed intracellulary. In the presence of 5 × 10 −3 m external choline- O -sulfate, the mycelia accumulated choline- O -sulfate to an apparent intracellular concentration of 0.075 m by 3 hr. Transport was unidirectional. No efflux or exchange of 35 S-choline- O -sulfate was observed when preloaded mycelia were suspended in buffer alone or in buffer containing a large excess of unlabeled choline- O -sulfate. (viii) The specific transport activity of the mycelium depended on the sulfur source used for growth. (ix) Sulfur starvation of sulfur-sufficient mycelium resulted in an increase in the specific transport activity of the mycelium. This increase was prevented by cycloheximide, occurred only when a metabolizable carbon source was present, and resulted from an increase in the Vmax of the permease, rather than from a decrease in Km . The increase could be partially reversed by refeeding the mycelia with unlabeled choline- O -sulfate, sulfide, sulfite, l -homocysteine, l -cysteine, or compounds easily converted to cysteine. The results strongly suggested that the choline- O -sulfate permease is regulated primarily by repression-derepression, but that intracellular choline- O -sulfate and cysteine can act as feedback inhibitors.