Cattle exposed to low doses of an Alberta crude oil, Pembina Cardium crude oil (PCCO), or a winter diesel oil no. 2 (WDO-2) were assessed for their biochemical activities in polymorphonuclear leukocyte (PMNL) cells (mainly neutrophils). The study used a randomized block design containing five treatment groups (8 animals/group). The animals were dosed per gavage with the test substance on study days 0, 14, 28, and 42. The dosages given (on per kg body weight) were: Group 1 (control), 10 mL/kg of potable water; Group 2, 5 mL/kg WDO-2; Group 3, 2.5 mL/kg PCCO; Group 4, 5 mL/kg PCCO; and Group 5, 10 mL/kg PCCO. Blood was collected at the specified intervals during the pre- and post-exposure periods, and the biochemical activities of isolated PMNL were analyzed. Cattle groups exposed to WDO-2 and PCCO showed moderate and statistically significant reductions (p < 0.01) in the activities of (1) phorbol myristate acetate (PMA) stimulated cellular respiration (respiratory burst), (2) NADPH-oxidase (PMA-stimulated production of superoxide anion), (3) myeloperoxidase, and (4) n-acetylglucosidase as compared to the control group. These biochemical parameters also showed statistically significant (p < 0.01) dose-related periodic (study day) trends. In general, these biochemical activities were decreased after each dosing; however, they subsequently recovered to near the pre-dosing levels. Such a biochemical response in PMNL provides a valuable biological tool to follow exposure effects in cattle accidentally exposed to low doses of petroleum hydrocarbons.