We studied the efficacy of the tris-glycinatocobaltate(II) complex ([Co(gly)(3)]) as a shift reagent (SR) for chloride by Cl-35 NMR spectroscopy and compared to that of Co-(aq)(2-). Due to the relatively low thermodynamic stability of [Co(gly)(3)](-), a 1:3 Co(II)/gly stoichiometric solution at physiological pH is approximately a 2:1 mixture of [Co(gly)(2)-(H2O)(2)] and [Co(gly)(H2O)(4)](+). This SR was found to be stable up to higher pH values than Co-(aq)(2+), better preventing Co(OH)(2) formation at alkaline pH. No significant differences in the Cl-35(-) NMR chemical shift induced by Co(II)/gly or Co-(aq)(2+) were observed in the presence of physiological concentrations of either Ca2+, or Mg2+, or of either Na+ or K+. Although Co-(aq)(2+) was almost twice as effective as Co(II)/gly in shifting the Cl-35 NMR resonance at the same high rho ([SR]/[Cl-]) value and low ionic strength, Co-(aq)(2+) showed a significant decrease (p < 0.05) in the Cl-35(-) chemical shift at higher ionic strength. Line widths at half-height were significantly (p < 0.05) less for Co(II)/gly than for Co-(aq)(2+) at rho values in the range 0.066-0.40. Intracellular chloride was clearly detectable by Cl-31 NMR spectroscopy in human skin fibroblast cells suspended in medium containing 40 mM Co(II)/gly SR. We determined that, although Co-(aq)(2+) provides a larger shift than Co(II)/gly at the same rho value, there are significant advantages for using Co(II)/gly, such as pH stability. ionic strength independent chemical shifts, narrow Cl-35(-) NMR resonances, and reduced cellular toxicity, as a SR in biological systems.