This publication analyzes different preparations of recombinant human growth hormone (rhGH) by hydrophobic-interaction chromatography (HIC). The effect of temperature on the separation was investigated as well as a series of commercially available HIC columns (TSK-phenyl-5PW, TSK-ether-PW, Beckman CAA-HIC and polypropyl A). The TSK-ether column gave the best results in the analysis of rhGH samples at different temperatures, as well as allowing an efficient separation of methionyl-hGH from rhGH. The TSK-phenyl column can be effectively used in the examination of different human growth hormone variants. The details of sample preparation have been demonstrated to be important in HIC analysis of hGH on the TSK-ether-5PW column. Injection volume and the solvent used to dissolve the protein sample are both crucial factors in this analysis. Also protein aggregation may play a role in these observations. The effect of temperature, protein concentration and spectroscopic data on the eluted protein suggest, however, that aggregation is not the cause of frontal peaks.