Clostridium perfringens and especially its spores, which are more tolerant to various physiochemical effects than the other fecal indicator bacteria, could serve as a useful indicator in ecosystems with stress factors. In order to determine the numbers of Clostridium perfringens in waters of rural North-West Greece, we utilised a new medium, lactose-sulfite (LS) broth, suggested for rapid enumeration and identification of Clostridium perfringens without the necessity for further confirmatory tests. Membrane filtration equipment was used on site. All samples were alternatively passed through two membrane filters, one (20-25 microns pore size) was used for retention of the abundant phytoplankton and the other (porosity 0.45 micron) for Clostridium perfringens. Membranes were placed into the first tube of 10-fold dilutions from 10(1) to 10(4) and incubated aerobically in a water-bath at 46 degrees C for 24 h. The numbers of Clostridium perfringens revealed fluctuations depending on the sampling site. It was significantly more common in the vicinity of industrial or domestic activities. LS broth not only allows the detection of small numbers of Clostridium perfringens but also permits rapid detection within 24 h. We propose the medium and the procedures described in this paper as a reliable technique for estimation of Clostridium perfringens as indicator of fecal contamination in waters.