Pulsed light (PL) is a technology that uses short, high-energy pulses of UV-rich broad spectrum light to inactivate a range of pathogenic and spoilage microorganisms in foods and on food contact surfaces. Microbial inactivation is directly related to the energy dose (fluence) received by the target microbes. Since fluence decays away from the lamp source due to light absorption and scattering phenomena, it is necessary to accurately quantify the fluence received locally within a substrate in order to design uniform antimicrobial PL treatments. The main objective of this work was to quantify and map the spatial distribution of both total and UV fluence both in air and in liquid substrates with different optical properties. Butterfield's phosphate buffer (BPB), trypticase soy broth (TSB), and apple juice (clarified apple cider) were used as substrates and Listeria innocua was used as the challenge microorganism. A pyroelectric head, and a UV-VIS-NIR spectrophotometer connected to a 1000 gm optical fiber, were used to measure the fluence in air and the three liquid substrates. Fluence was measured at incremental distances from the lamp, along the x-, y-and z-axes. As expected, fluence decreased with increasing distance from the lamp, in all three directions. A 3-parameter Gaussian model described well the spatial distribution of fluence, both in air and in the liquid substrates. Overall, this study emphasizes the fact that substrate-light interactions affect the spatial distribution of fluence within a substrate, and these non-uniformities need to be taken into account when developing commercial applications of PL (C) 2010 Elsevier Ltd. All rights reserved.