New estimation methods of bacterial cell concentration in samples by measurement of the increase in bacterial adenosine-triphosphate (ATP) content during incubation using a conventional firefly luminometer were established. When an Escherichia coli cell suspension was incubated in nutrient broth, the increase in the ATP content of the suspension during the incubation period followed a sigmoidal curve. The increase ratio of the ATP content of the suspension at a given period of incubation (5 hours in this study) to the initial ATP content was greater at higher initial cell concentrations. With this relationship, the initial cell concentration of a test suspension could be predicted from the measured ratio; this was called the end point method. On the other hand, the lag period in the ATP increase curve was longer at lower initial cell concentrations. A highly linear relationship was observed between the lag period and the logarithm of the initial cell concentrations. Based on this relationship, a delay method was developed for prediction. The two relationships were also observed for bacterial suspensions of Klebsiella sp., Staphylococcus aureus, Bacillus subtilis, and Pseudomonas sp. These results suggested that the two methods have the potential to estimate the bacterial cell concentration of a sample suspension.