Cultures of Mycoplasma hyopneumoniae and M. flocculare in Friis' broth grew faster and to higher titers in air than in 8% CO2; cultures in air grew better when shaken than when stationary. Under the optimal conditions, both species have generation times of about 10 h and achieve maximum titers of at least 10(9) organisms per ml. Maximum growth was reached near pH 7.0, before the phenol red indicator had noticeably changed colour. Changes in growth were readily detected by an ATP assay based upon the luciferin-luciferase reaction. Concentrations of ATP fell rapidly after peak growth. Although the addition of 27 mM glucose to the medium did not change the pattern of growth and gas chromatography gave no evidence of the production of volatile or non-volatile end-products, washed harvested cells of both species metabolized [14C]-glucose. The addition of 29 mM arginine to the medium inhibited growth.