The phosphane Cu(I) complex [Cu(thp)₄][PF₆], 1 (thp = tris(hydroxymethyl)phosphane) shows notable in vitro antitumour activity against a wide range of solid tumours. Uptake experiments performed in 1-treated colon cancer cells by atomic absorption spectrometry, reveal that the antiproliferative activity is consistent with the intracellular copper content. The solution chemistry of this agent, investigated by means of X-ray Absorption Spectroscopy and spectrophotometric titrations in aqueous media, indicates that 1 is labile giving coordinative unsaturated [Cu(thp)_n]⁺ species (n = 3 and 2) at micromolar concentrations. [Cu(thp)_n]⁺ are reactive species that yield the mixed-ligand complex [Cu(thp)₂(BCS)]⁻ (BCS: bathocuproinedisulphonate^(2-)) upon interaction with N,N-diimine. Analogously, [Cu(thp)_n]⁺ interact with the methionine-rich peptide sequence (Ac-MMMMPMTFK-NH₂; Pep1), relevant in the recruiting of physiological copper, giving [Cu(thp)(Pep1)]⁺ and [Cu(Pep1)]⁺ species. The formation of these adducts was assessed by electrospray mass spectrometry in the positive ion mode and validated by density functional theory investigations. The possibility to trans-chelate Cu(I) from pure inorganic [Cu(thp)_n]⁺ assemblies into more physiological adducts represents a pathway that complex 1 might follow during the internalization process into cancer cells.