Hexafluoroisopropanol (HFIP) is a nonpolar organic solvent that is often used to prepare β-amyloid peptide (Aβ) samples. In this work, we compare the effects of two different species derived from synthetic Aβ1–42 and prepared without HFIP (Aβ) or using HFIP (Aβ/HFIP) on the glycine-activated chloride current (IGly). The experiments were conducted on the pyramidal neurons isolated from CA3 region of rat hippocampus. Transmembrane currents were recorded using a conventional patch-clamp technique in the whole-cell configuration. The IGly was induced by a step application of the agonist for 600 ms through glass capillary. Aβ or Aβ/HFIP was coapplied with glycine. The effects of the two species of the peptide have similar and distinctive features. Both substances caused a reduction in the peak amplitude and an acceleration of desensitization of the IGly. At the same time, the effect of Aβ/HFIP was found to develop and recover more slowly and required several repeated applications for its saturation (use dependence). The effect of Aβ/HFIP was voltage independent and equally pronounced at negative and positive membrane potentials. First, our results confirm that HFIP pretreatment may influence the properties of Aβ. Second, new information on the glycine receptor ability to interact with drugs in use-dependent mode was obtained.