Cows (n = 12/genotype) from unselected (stable milk yield since 1964, UH) and contemporary (CH) Holsteins that differed by more than 4,500 kg milk/305 d were fed the same diet ad lib and housed together for more than 4 mo before being blocked (2/genotype) by DIM and randomly assigned within genotype to receive saline or 0.25 μg LPS (Escherichia coli 055:B5) per kg BW. Cows were synchronized to be at d 8 of their estrous cycle for the first challenge (C1) at 70–84 d in milk. Jugular catheters were implanted 24 h before C1. Blood samples were collected at −1, −0.5, 0, 1, 2, 3, 4, 6, 8, 12, and 24 h relative to treatment administration and plasma harvested. Body temperatures (BT) were determined at these times and at 5 and 7 h. Liver biopsies and blood for flow cytometry and hemogram assays were obtained at 0, 4, and 24 h. A second identical challenge (C2) and sampling was conducted 4 d later. Data were analyzed by repeated measures using PROC MIXED (SAS). Means differed when P < 0.05. Pre-challenge glucose and IGF-1 were greater (P < 0.01) and BT was less (P < 0.01) in UH than CH. Glucose response to LPS was greater (P < 0.01) in UH than CH, but IGF-1 and BT response was similar in both genotypes. TNFα and cortisol response to LPS was greater during C1 than C2 (P < 0.02). TNFα response to LPS was greater (P < 0.05) in UH than CH in C1 but similar in C2. Cortisol response to LPS increased in both genotypes but returned to baseline earlier in CH than UH (P < 0.05). LPS decreased white blood cell count (P < 0.01) but response did not differ between genotypes or challenge. Neutrophil oxidative burst was greater (P < 0.05) and phagocytic capacity tended (P = 0.07) to be greater in UH than CH. CD11b expression increased (P < 0.05) in response to LPS at 4h, was less in CH than UH at 24h and did not differ between C1 and C2. L-selectin decreased in response to LPS at 4hr but did not differ between challenge or genotype. Results indicate that genotype affects bovine response to LPS and this effect differs among the response variables assessed.