Two groups of ten polyurethane substrates of different sizes ranging from 45 mm3 to 25,600 mm3 were set out in Douglas Lake, Michigan. Substrates were sampled for fresh-water protozoa by squeezing them to extract a sufficient volume of water for examination. The number of fresh-water protozoan species in samples taken from the substrates over a six-week period was determined, and comparisons were made between the number of species in substrates with different sizes and between the number of species for each substrate at different sampling periods. The number of species found per substrate varied from zero to 32 with an increase in number of species over time. The maximum number of species recorded for each substrate was found with the 4th to 6th weeks of sampling. For the substrates that peaked early (at 4 weeks) the number of species remained nearly constant or decreased slightly after the peak. Results indicate that within a minimal size range, substrate volume affects the number of fresh-water protozoan species. Smaller substrates generally had fewer species than the larger substrates, and a linear relationship was found between log volume and number of species.