Question In many industrialized countries biodiversity is declining. Although changes in species composition and species richness have been documented for many individual systems, little long-term research has been done on a regional scale. We compared the temporal patterns of plant diversity over the last 30 yr in agricultural grasslands and nature reserves. Location Grasslands scattered over the entire province of South-Holland, the Netherlands. Methods Using data from 403 permanent plots over a period of 30 yr, we analysed temporal trends in species richness, floristic value and indicated nutrient richness and compared these trends between agricultural grasslands and grasslands in nature reserves. Results Expressed as 30-yr averages, species richness and floristic value were much higher in nature reserve grasslands than in agricultural grasslands (an increase of 87% and 78%, respectively), while indicated nutrient richness (determined using indicator values for soil fertility of the species present) was 31% lower. During this 30-yr period, species richness and floristic value significantly increased in nature reserve grasslands (0.186 species.yr-1). For agricultural grasslands, however, no significant changes could be detected. Restricting the analysis to those plots that were already studied before 1990 did reveal significant declines in the agricultural grasslands, indicating a past decrease in species richness that has probably levelled off after 1990. Conclusions These findings imply that the floristic gap between agricultural grasslands and grasslands in nature reserves is widening every year. This may have consequences for conservation efforts in both nature reserves and agricultural grasslands. The challenge is to enhance the interaction between nature reserves and agricultural grasslands.