Cysteine remains the preferred supplement for cultivation of Cys- auxotrophs in vitro. Methionine, which reduced cysteine requirements, and branched-chain amino acids, which decreased cysteine toxicity, were identified as the components of casein hydrolysate responsible for growth enhancement by this additive. Glutathione and DL-homocysteine can be substituted for cysteine. Accumulation of these compounds in patients with renal impairment may favor selection of Cys- strains in vivo.