Ten isolates of Mycoplasma synoviae (MS) from air sacs and joints of chickens were propagated in a medium enriched with diphosphopyridine nucleotide (NAD), cysteine, and glucose. Yields of antigen were 1.4 to 3.3% of the total medium. Three antigens did not agglutinate with all heterologous antisera. In 12 different trials with the same isolate, antigen production varied markedly, ranging from 1.6 to 3.9%. The several antigens tested were essentially the same in sensitivity. Experiments were conducted with the WVU 1853 isolate to determine growth requirements, with the following results: agar, glucose, cysteine, and additional NAD added to the basal medium did not enhance growth. Nicotinamide replaced NAD in the basal medium for the growth of a strain of MS that had been serially passed in broth enriched with NAD.