Variations in ATP content in three ground beef spoilage bacteria, Lactobacillus brevis, Lactobacillus jensenii, and Pseudomonas sp. were investigated using the bioluminescence (luciferin-luciferase) assay. Environmental factors (temperature, atmosphere, pH, aeration, and phase of growth), as well as differences among genera and species, were studied in relation to their effect on cellular ATP. Variations for each of the environmental factors and bacteria were shown statistically to be significantly different at the 0.05 level. The mean ATP/cell for each of the bacteria was 2.71 fg/cell (L. brevis), 2.20 fg/cell (L. jensenii), and 1.36 fg/cell (Pseudomonas sp.). For all three bacteria, ATP/cell was lower and more stable throughout the culture's growth cycle at 3°C or in N₂. In general, ATP/cell increases from a lowest value in lag phase to a highest value in stationary phase. The effect of sonication on ATP/cell was tested for each bacterium at one set of factors. Sonication studies showed that L. brevis cells were clumping, especially in aged cultures. After sonication, ATP/cell remained relatively constant from lag through stationary phase. L. jensenii showed no signs of clumping and ATP/cell increased as the culture aged. Sonication had a lethal effect on the Pseudomonas. Thus the ATP/cell for Pseudomonas increased dramatically as the culture aged. Guidelines concerning temperature, assumed ATP content for major contaminants, and sample handling must be followed in order to use the bioluminescence assay to estimate biomass in foods.