Nutrient limitation plays an important role in shaping community structure and ecosystem processes in aquatic environments. Many types of nutrient diffusing substrata (NDS) have been used to estimate nutrient limitation in lotic systems. However, whether these various NDS methods produce comparable results is unknown. We evaluated the 3 most commonly used NDS methods—clay pots, plastic cups, and periphytometers—in a single stream to determine if they gave qualitatively similar results. We also examined the effects of initial nutrient ratios on diffusion rates in all 3 types of NDS and periphyton stoichiometry on clay pots. The largest response in chlorophyll a biomass consistently occurred on substrata that simultaneously diffused both inorganic N and P. However, each NDS method produced a significantly different picture of limitation. Clay pots showed that primary producers were colimited by N and P, plastic cups showed primary limitation by N and secondary limitation by P, and periphytometers showed primary limitation by P and secondary limitation by N. Nutrient diffusion rates were very different among methods. Effects of different N∶P ratios were only seen in clay pots. When N∶P was 16∶1, chlorophyll a biomass was low. When N∶P was 1∶1, periphyton had greater %C and %P and low C∶P and N∶P. Our results indicate that further research is required to clarify methodological differences between the types of NDS. Until such discrepancies are addressed, the results obtained with NDS methods should be interpreted with caution.