<jats:p> Mechanisms for regulation of puromycin biosynthesis in <jats:italic>Streptomyces alboniger</jats:italic> were studied by measuring the levels of <jats:italic>S</jats:italic> -adenosyl- <jats:sc>l</jats:sc> -methionine: <jats:italic>O</jats:italic> -demethylpuromycin <jats:italic>O</jats:italic> -methyltransferase. The enzyme was released in soluble form from mycelia by 3 to 5 min of sonication at 4 C. Maximal specific activities of 0.7 and 0.1 nmol/min per mg of protein were found in cells grown in corn steep liquor-corn starch and Hickey-Tresner media, respectively. In both media, the <jats:italic>O</jats:italic> -methyltransferase activity rose from low levels to a maximum during midlogarithmic growth and then declined or disappeared completely (in Hickey-Tresner medium) during stationary phase. Either glucose (1%) or ethidium bromide (5 μM) reduced <jats:italic>O</jats:italic> -methyltransferase formation to very low levels with no effect on overall growth. Complete glucose repression of antibiotic formation occurred on agar. Cells grown in the presence of ethidium bromide continued to produce low enzyme levels after regrowth in the absence of dye, but formed normal amounts of puromycin on Hickey-Tresner agar. The <jats:italic>O</jats:italic> -methyltransferase, either crude or purified, was rapidly inactivated at 37 C. Each substrate alone, or both together at lower concentrations, protected against this loss of activity. Puromycin inhibited the transferase. Regulation of <jats:italic>O</jats:italic> -methyltransferase synthesis in <jats:italic>S. alboniger</jats:italic> includes (i) induction early in growth that is susceptible to catabolite repression and differential inhibition by ethidium bromide, and (ii) protection of the enzyme from inactivation by increased intracellular levels of its substrates. The <jats:italic>O</jats:italic> -methyltransferase was purified 30- to 40-fold by a combination of protamine sulfate precipitation, ammonium sulfate fractionation, adsorption and gradient salt elution from diethylaminoethyl-cellulose and Sephadex G-200 gel filtration. The enzyme was very unstable, even at low temperatures, upon purification beyond the salt fractionation step, but was stabilized by the addition of <jats:italic>S</jats:italic> -adenosyl- <jats:sc>l</jats:sc> -methionine during later stages of purification.