Mycoplasma fermentans organisms in medium containing 20% horse serum multiplied to 10(7)-10(10) ccu/ml within 5 days at 37 degrees C and were dead usually after 9 days. There was no growth in medium without serum, nor in such medium with the addition of cholesterol or palmitic acid or both, but in some experiments addition of bovine plasma albumin (BPA) increased the number of organisms by up to 1000-fold and some remained viable for up to 84 days. BPA and cholesterol or BPA, palmitic acid and cholesterol more often enhanced growth, in terms of the maximum number of organisms and their survival, than did the addition of BPA alone. The maximum number of organisms in such supplemented serum-free media was usually at least ten-fold less than in medium with horse serum, but some organisms remained viable for up to 131 days. Survival of Ureaplasma urealyticum was also longer in the supplemented serum-free medium than in standard horse-serum medium. The possible factors affecting persistence of mycoplasmas in culture are discussed in relation to these observations.