The complex formation between silver(I) and cysteine (H2Cys), penicillamine (H2Pen) or glutathione (H3Glu) in alkaline aqueous solution was examined using extended X-ray absorption fine structure (EXAFS) and 109Ag NMR spectroscopic techniques. The complexes formed in 0.1 mol·dm−3 Ag(I) solutions with cysteine and penicillamine were investigated for ligand/Ag(I) (L/Ag) mole ratios increasing from 2.0 to 10.0. For the series of cysteine solutions (pH 10 - 11) a mean Ag-S bond distance 2.45 ± 0.02 Å consistently emerged, while for penicillamine (pH 9) the average Ag-S bond distance gradually increased from 2.40 to 2.44 ± 0.02 Å. EXAFS and 109Ag NMR spectra of a concentrated Ag(I)-cysteine solution (CAg(I) = 0.8 mol·dm−3, L/Ag = 2.2) showed the mean Ag-S bond distance 2.47 ± 0.02 Å and δ(109Ag) = 1103 ppm, consistent with prevailing, partially oligomeric AgS3 coordinated species, while for penicillamine (CAg(I) = 0.5 mol·dm−3, L/Ag = 2.0) the mean Ag-S bond distance 2.40 ± 0.02 Å and δ(109Ag) = 922 ppm indicate that mononuclear AgS2 coordinated complexes dominate. For Ag(I)-glutathione solutions (CAg(I) = 0.01 mol·dm−3, pH ~ 11), mononuclear AgS2 coordinated species with the mean Ag-S bond distance 2.36 ± 0.02 Å dominate for L/Ag mole ratios from 2.0 to 10.0. The crystal structure of the silver(I)-cysteine compound (NH4)Ag2(HCys)(Cys)·H2O (1) precipitating at pH ~ 10 was solved and showed a layer structure with both AgS3 and AgS3N coordination to the cysteinate ligands. A redetermination of the crystal structure of Ag(HPen)·H2O (2) confirmed the proposed digonal AgS2 coordination environment to bridging thiolate sulfur atoms in polymeric intertwining chains forming a double helix. A survey of Ag-S bond distances for crystalline Ag(I) complexes with S-donor ligands in different AgS2, AgS2(O/N) and AgS3 coordination environments was used, together with a survey of 109Ag NMR chemical shifts, to assist assignments of the Ag(I) coordination in solution.