Growth of T-strain mycoplasmas in standard broth cultures has been characterized by rapid growth to peak titers of 10(6) to 10(7) color change units/ml at 20 to 24 hr, followed by a sharp decline in the viability over the next few hours. In a vacuum-flow (V-F) system utilizing negative pressure equal to 10 cm of water and air flow on the surface of broth, T strains were seen to enter the stationary phase of growth after 24 hr, and viability titers after 72 hr were >/= 10(4) color change units/ml. Maintenance of organisms in the stationary phase required both vacuum and air flow, a medium depth of 1 mm, and 1% urea in the broth. Concentration of ammonia in broth cultures under V-F remained below 200 mug/ml during 72 hr of observation. Ammonia levels in standard broth cultures exceeded 200 mug/ml after 24 hr, coincident with the decline in viability. Air passed over the medium surface in the V-F system contained large amounts of ammonia; the amount increased during each succeeding 24-hr period of observation.