Periphyton nutrient limitation was assessed in Lake Okeechobee, a large, shallow, eutrophic lake in the southeastern U.S.A. Nutrient assays were performed to determine if the same nutrients that limit phytoplankton also limit periphyton growth in the lake. Nutrient diffusing clay substrates containing agar spiked with nitrogen, phosphorus, or both, along with nutrient-free controls, were incubated at four sites in the lake. Three sites were located in a pelagic-littoral interface (ecotone) and one site was located in the interior littoral region. Incubations lasted for 20-26 days, and were repeated on a quarterly basis between 1996 and 1997, to incorporate seasonal variability into the experimental design. The physical and chemical conditions at each site also were measured. Periphyton biomass (chlorophyll a and ash-free dry mass) was highest at the littoral and northern ecotone sites. At the littoral site, nitrogen limited biomass in four of five incubations, although the largest biomass differences between the treatments and controls (less than or equal to3 mug cm(-2) as chl) were probably not ecologically significant. Periphyton biomass at the western and southern ecotone sites was low compared to the other two sites. Increases in water column depth and associated declines in light penetration strongly correlated with periphyton growth and suggested that they may have limited growth most often at all three ecotone sites. Nitrogen also was found to limit periphyton growth approximately 20% of the time at the ecotone sites and phosphorus was found to limit growth once at the west site.