We have studied the conformational behavior of α-lactalbumin (α-LACT) in hydrophobic interaction chromatography (HIC). Retention characterization in terms of Z (Slope of plot 1n k′ vs. 1n ø B, where k′ is the capacity factor and Ø B is the volume fraction of mobile phase B) has been explored, and the relationship of Z to other slopes, such as S (slope of the plot of 1n k′ vs. ø Bf) has been derived. The reason for the sensitivity of Z to conformational change are discussed. The enhanced broadening of α-LACT in a temparature transition region of conformational change has been studied by spectral analysis using on-line photodiode array detection. The influence of Ca 2+ and Mg 2+ addition to the mobile phase is further explored. Since α-LACT is a calcium binding protein, addition of this metal leads to stabilization, i.e higher column temperatures are required for conformational change. On the other hand, addition of Mg 2+ appears to destabilize the protein. We have explored the use of a more hydrophobic support, C 2-(ethyl) ether phase, for the elution of α-LACT. In this case, two widely separated peaks ae observed. By spectral analysis the first peak is shown to be native and the later eluted, broad second peak to ber an unfolded mixture of species. As previously observed in reversed-phase liquid chromatography, the second peak grows at the expense of the first, as the column temperature is raised. The second peak also grows as the contact time of the protein with the surface increases. This behavior can be ascribed to the conformational change of α-LACT in the column, the late eluted species under the second peak binding significantly more strongly to the phase than the native peak. Reinjection of the late eluted fraction reveals the reformation of the native species takes place in solution within 30 min. As before, addition of Ca 2+ reduces the extent of unfolding under any specific condition. These results add further to our understand and ability to control conformational changes in high-performance liquid chromatography.