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The transition of grasslands to forests influences many ecosystem processes, including water and temperature regimes and the cycling of nutrients. Different components of the carbon biogeochemical cycle respond strongly to woody plant encroachment; as a consequence, the carbon balance of the invaded grasslands can change markedly. In our research, we studied the response of soil respiration (RS) to natural succession of calcareous grassland. We established two research sites, called grassland and invaded site, at each of which eddy flux measurement were also performed. Within these sites, triplicate plots were fenced for soil flux measurements. At the invaded site, measurements were performed for forest patches and grassy spaces separately. Soil respiration was strongly dependent on temperature and reached 8–12 μmol CO2 m−2 s−1 in mid-summer; it was greater at the grassland than at the invaded site. RS dependence on temperature and soil water content was similar between the different vegetation covers (grassland, gaps and forest patches). At a reference temperature of 10°C, the average RS was 2.71 μmol CO2 m−2 s−1. The annual sums of RS were also similar between years and sites: 1345 ± 47 (2009) and 1150 ± 37 g C m−2 year−1 (2010) for grassland and 1324 ± 26 (2009) and 1268 ± 26 g C m−2 year−1 (2010) for the invaded site, which is at the upper range of the values reported in the literature. Cumulative RS peaked in July, with about 200 g C m−2. Large mid-summer RS rates rely on strong biological activity supported by high, but non-extreme soil temperatures and by regular summer precipitation. A coupling of photosynthesis and RS was revealed by a 24-hour measurement, which showed asymmetrical clockwise hysteresis patterns.