Little is known about the long-term effects of mineral N, P and K application on the nutritional status of mown alluvial grasslands. We asked how long-term fertilizer application affected soil chemical properties, plant species composition, herbage production, nutrient concentrations in soils and plants and balance of nutrients. Six treatments (control, PK, N50PK, N100PK, N150PK and N200PK) were investigated at the Černíkovice Experiment (Czech Republic) established in 1966 on an Alopecurus pratensis meadow, using annual application rates of 50, 100, 150 and 200 kg N, 40 kg P and 100 kg K ha−1. Data were collected and analysed for 2007, 2008 and 2009. Although fertilizers had been applied over 45 years, differences in soil chemical properties between fertilization treatments were small. The legumes Lathyrus pratensis and Trifolium repens responded highly positively to PK application, and tall grasses, A. pratensis in particular, to NPK application. Herbage quality was high in terms of content of major nutrients, and its chemical properties varied considerably between treatments, cuts and years. Mean annual herbage yield ranged from 6·1 in the control to 9·7 t ha−1 in the N200PK treatment. Herbage production was N-limited in 2007 and 2009, but not in 2008. Seasonal N agronomical efficiency ranged from 4·2 to 22·9 kg of DM herbage per kg of applied N. The herbage N:P and N:K ratios did not reflect the actual response of herbage production to N application. A negative balance between N applied and N removed in harvested herbage was recorded in all treatments. We concluded that in highly productive alluvial grasslands, mineral-rich soils can respond weakly to N, P and K application, fertilizer application modifies plant species composition and herbage production is not N-limited in all years. Nutrient ratios must be interpreted with caution for the estimation of nutrient limitation.