During an 8-year period, a study was conducted in a large dairy in the Sacramento Valley of California, to define factors associated with the introduction and spread of mycoplasmal mastitis. To identify cows in which mycoplasmal infection appeared in 22 of 25 (88%) periods of new infection, milk samples were collected weekly from freshened cows and from hospitalized cows with clinical mastitis. The disease first appeared in freshened cows in at least 36% of the periods of new cases of mycoplasmal mastitis, which indicated that special attention must be paid to freshening cows in an attempt to control spread of the disease. New cases of mycoplasmal mastitis were recorded more often in January through April than during the rest of the year. Rates of mastitis infection caused by other pathogens (contagious and environmental organisms) increased during months when new cases of mycoplasmal mastitis were recorded. Sanitation on the farm during udder infusion and milking was a major factor in controlling the introduction and spread of mycoplasmal mastitis.