In most ripened cheeses, bacteria are responsible for the ripening process. Immobilized in the cheese matrix, they grow as colonies. Therefore, their distribution as well as the distance between them are of major importance for ripening steps since metabolites diffuse within the cheese matrix. No data are available to date about the spatial distribution of bacterial colonies in cheese. This is the first study to model the distribution of bacterial colonies in a food-type matrix using nondestructive techniques. We compared (i) the mean theoretical three-dimensional (3D) distances between colonies calculated on the basis of inoculation levels and considering colony distribution to be random and (ii) experimental measurements using confocal microscopy photographs of fluorescent colonies of a Lactococcus lactis strain producing green fluorescent protein (GFP) inoculated, at different levels, into a model cheese made by ultrafiltration (UF). Enumerations showed that the final numbers of cells were identical whatever the inoculation level (10(4) to 10(7) CFU/g). Bacterial colonies were shown to be randomly distributed, fitting Poisson's model. The initial inoculation level strongly influenced the mean distances between colonies (from 25 μm to 250 μm) and also their mean diameters. The lower the inoculation level, the larger the colonies were and the further away from each other. Multiplying the inoculation level by 50 multiplied the interfacial area of exchange with the cheese matrix by 7 for the same cell biomass. We finally suggested that final cell numbers should be discussed together with inoculation levels to take into account the distribution and, consequently, the interfacial area of colonies, which can have a significant influence on the cheese-ripening process on a microscopic scale.