The development of a simple, reliable method for determination of detergent micelle aggregation number that relies solely on measurement of steady-state fluorescence quenching is presented. The degree of steady-state fluorescence quenching of a micelle-solubilized fluorophore (pyrene) by a quencher that partitions greatly into the micelles (coumarin 153) is dependent on the micelle concentration, which can therefore be determined. The aggregation number is calculated as the micelle concentration/detergent monomer concentration (the total detergent concentration above the critical micelle concentration). For the determination to be accurate, the partition coefficient of the quencher into the micelle phase is determined and used to calculate the micellar concentration of quencher. Also, the quenching of pyrene by a coumarin 153 molecule within the same micelle must be complete, and this was confirmed by time-resolved fluorescence measurements. Aggregation numbers were determined for one cationic and several nonionic detergents and were found to be consistent with literature values. The approach presented is an improvement on a previous luminescence quenching technique (Turro, N.J., and A. Yekta. 1978. J. Am. Chem. Soc. 100:5951-5952) and can be used on cationic, anionic, and nonionic detergents with micelles ranging greatly in size and under varying conditions, such as detergent concentration, ionic strength, or temperature.