This study evaluated relationships among intramammary challenge with Staphylococcus aureus, SCC, and susceptibility to infection and determined the genetic contribution to susceptibility of infection with special emphasis on bovine lymphocyte antigens. Intramammary challenge with S. aureus Newbould 305 was used as a model for natural infection. A total of 124 cows were challenged in all quarters with 600 cfu of S. aureus Newbould 305 in 2 ml of PBS solution. Quarter milk samples for bacteriology and SCC were taken at three times both before and after challenge. Lymphocytes were separated from whole blood and tested with a standard microlymphocytotoxicity test to determine the presence of bovine lymphocyte antigens class I alleles. One hundred and thirteen cows were negative for S. aureus at challenge, and 11 cows exhibited positive S. aureus counts in one or more quarters at challenge. Fifty-three percent of quarters from the 124 cows challenged became infected after challenge. Nineteen cows had no quarters infected; 19 cows, one; 26 cows, two; 25 cows, three; and 24 cows had four quarters infected after challenge. Average SCC before challenge was 181,000 and after challenge was 758,600. The SCC prior to challenge of cows that resisted infection were higher than prior SCC of cows that became infected (282,000 and 91,000, respectively). The reverse was true after challenge. Presence of the bovine lymphocyte antigens class I allele CA42, instead of EU28 (based on gene substitution), increased susceptibility to S. aureus infection. Heritability estimates of S. aureus susceptibility after experimental challenge were low and unstable.