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The aim of this study was to determine the effects of cultivar, nitrogen (N) rate and harvest time on the content of water-soluble carbohydrates (WSC), crude protein (CP) and the WSC:CP ratio in the aboveground biomass of perennial ryegrass (Lolium perenne L.). A small-area field experiment was conducted in the Agricultural Experiment Station in Tomaszkowo (53°42′40.8″ N 20°26′04.7″ E, north-eastern Poland). Data were presented for three years of full utilization (2013–2015). The experimental factors were as follows: (i) perennial ryegrass cultivar: Bajka and Baronka, (ii) N rate (kg ha−1): 0, 120, and 240, and (iii) harvest time: 8.00–10.00 a.m., 12.00–2.00 p.m., and 4.00–6.00 p.m. The tetraploid cultivar Baronka had higher WSC content and lower CP content on a dry matter (DM) basis than the diploid cultivar Bajka (by approx. 3% on average). An increase in N fertilizer rate to 240 kg ha−1 contributed to a decrease in WSC concentrations by 23% in cv. Bajka and by 19% in cv. Baronka, and to an increase in the CP content of aboveground biomass by 32% and 23%, respectively. Both cultivars accumulated significantly higher WSC amounts when biomass was harvested at noon and in the afternoon than in the morning. The CP content of biomass was also affected by harvest time, and it was higher in cv. Bajka when harvesting was carried out at 12.00–2.00 p.m. and 4.00–6.00 p.m., and in cv. Baronka when harvesting was carried out at 4.00–6.00 p.m. The WSC:CP ratio in the biomass of the analyzed cultivars was within the optimal range for ruminants, and it was higher in cv. Baronka (1:1.11). Nitrogen applied at 240 kg ha−1 had a negative influence on the WSC:CP ratio, decreasing its value by around 36% (relative to the rate of 120 kg N ha−1) and around 35% (relative to the unfertilized treatment). In both perennial ryegrass cultivars, biomass harvested in the morning was characterized by the lowest WSC:CP ratio. [ABSTRACT FROM AUTHOR]