Head kidney leukocytes are central elements in a number of in vivo and in vitro assays elucidating innate and adaptive immune mechanisms in teleosts following stimulation with various antigens. These systems are sensitive to a number of factors affecting the outcome of the assays. The present work describes the importance of temperature, cell concentration, immunostimulant, exposure time and immune-modulatory molecules on the respiratory burst activity of rainbow trout head kidney leukocytes in vitro. Some variation in RBA was observed among individual fish. However, use of cells pooled from four individuals produced satisfactory results following exposure to PMA, zymosan and ß-glucan. Temperature was shown to have a significant effect on production of reactive radicals with highest activity in cells maintained at 15-20oC and reduced activity at extremes (1oC, 4oC and 30oC). When cells were tested for RBA at different time intervals following harvest, the highest RBA was recorded in freshly isolated head kidney cells (0 h) with a clear decline with increase in in vitro culture time. When cells were exposed to stimulants for different time period and RBA was measured, highest activity was recorded where cells were exposed for 2-4 h. Further increase in incubation time with stimulants lead to decrease in response. RBA was found to be positively correlated to the cell concentration. Highest activity was recorded using a concentration of 1 x 107 cells/ml although measurable activity was recorded from cells in a concentration of 1 x 104 cells/ml. Co-incubation of cells with inhibitory substances (DiMePE2, cortisol and SOD) decreased the RBA. It is concluded that several biotic and abiotic factors should be taken into account when conducting RBA assays with head kidney leukocytes for elucidation of rainbow trout immune responses.