In a 270-cow dairy herd, milk samples from all cows with acute mastitis were cultured during a 12-month period. Of 119 isolates (from 166 quarters), 49% were gram-positive organisms, 39% were gram-negative organisms, and 12% were yeasts. Of all samples from cows with acute mastitis, 40% failed to produce colonies on culture media. Recovery of the common gram-positive pathogens, Staphylococcus aureus (11% of isolations) and Streptococcus agalactiae (1% of isolations), was rather low. However, S epidermidis (14% of isolations) and streptococci other than Str agalactiae (9% of isolations) were recovered more frequently than expected, particularly in recently calved cows. Coliform organisms accounted for 35% of the total isolates and were the single most important cause of acute mastitis. Of 42 cows with coliform mastitis, 6 died despite intensive antibiotic and electrolyte therapy. There was no death loss among cows with mastitis caused by gram-positive organisms. Antimicrobial susceptibilities of 95 isolates to 11 antimicrobials was determined by standardized methods. Organisms were most often susceptible to chloramphenicol, cephalothin, and gentamicin. A treatment regimen consisting primarily of oxytetracycline and/or sulfonamides systemically in conjunction with intramammary infusions of cephapirin was evaluated. Treatment response in 37 quarters was determined by culturing before treatment and at 4 or more days after treatment. Of 23 quarters initially culture-positive, 22 were negative on reculture. Treatment response appeared to agree with the results of the susceptibility testing.