Many plug seedling growers complain about the inadequacy of substrate testing as a measure of nutritional status because results are too variable. We conducted two experiments to test a model system of sampling substrate at a set time after fertilization. Petunias ( Petunia×hybrida Hort. ex Vilm. var. multiflora `Primetime White') were grown in 288-cell plug trays. Six fertilizer regimes were used consisting of a factorial arrangement of three fertilizer cycles (at each, every other, and every third irrigation) and two leaching fractions (0% and 20%). Fertilizer or water was applied at 0900 HR daily, and then 24 hours later in Expt. 1, and 1 hour later in Expt. 2, substrate solutions were sampled and analyzed. Samples taken after waterings were used to assess the dilution and leaching effects of water on substrate nutrient concentrations. In Expt. 2, additional substrate samples were taken at various hours after fertilizing to test the effect of plant depletion of the substrate. Substrate nutrient concentration curves constructed from data drawn at a fixed time after fertilizations, but not after waterings, were logical and could be interpreted. When data from a fixed time after fertilizations and waterings were plotted together, the curves could not be interpreted. Data from samples taken at various hours after fertilization in Expt. 2 revealed large reductions in concentrations, often after only 4 hours. Overall, leaching and dilution effects from watering in combination with the increased time span from fertilizing to sampling resulted in nutrient concentrations that could not be interpreted. Substrate testing can be effective for plug seedling production, but samples need to be taken 1 to 2 hours after fertilizations.