Copper (Cu) based pesticides are widely used in viticulture and are permitted in organic viticulture. Due to its extensive long term use, Cu accumulates in vineyard soils and ecotoxicological implications are growing. In this study, the cycling of Cu based pesticides was investigated in vineyard environments using copper mass balance, electron paramagnetic resonance (EPR) spectroscopy and Cu isotope analyses. Different soils (i.e. vertic cambisol and calcaric cambisol) from the Soave vineyard (Italy) were studied. Kinetic extractions were performed on soil samples using Na3-citrate to assess the bioavailable Cu fraction. Results show that isotope ratios of pesticides depend on Cu speciation and their manufacturing date, covering a large range of isotope ratios (δ65Cu from −0.49 ± 0.05‰ to 0.89 ± 0.01‰) making it difficult to trace sources of Cu in soils. Mass balance calculations based on Ti as invariant element permitted to put in evidence large excess Cu stocks in both studied soils. Excess Cu is transported to depth with approximately the same apparent rate (0.0092 m yr− 1) in both soils, faster than formerly reported in literature. A substantial amount of Cu was missing from calcaric cambisols (6 to 48%) when compared to vertic cambisols, implying a relative loss of Cu from such soils via the soil solution. In bulk soils, there are slight but significant differences in mean Cu isotope ratios depending on soil type (δ65Cu from 0.28 vs 0.18‰ in vertic and calcaric soils respectively), illustrating the loss of heavy Cu from carbonated soils. EPR analysis confirms a difference in Cu speciation between vertic and carbonate-rich soils, indicating an influence of carbonates on Cu retention besides the role of Cu-organic matter interactions. Kinetic extractions showed that the bioavailable fraction displays isotopically heavier Cu isotopes signature than bulk soil, whatever the soil type.