Free-radical peroxidation of cholesterol results in, among others, another products of its oxidation, called oxysterols. Scientists are still more and more interested in oxysterols, because, like the products of polyunsaturated fatty acids, show a strong biological activity, which effects physiology, pathology and pharmacology. The aim of the work was to investigate whether pre-pregnancy diabetes effects cholesterol oxidation process studied on the basis of the concentration of the chosen oxysterols. The chosen oxysterols were determined in 45 patients suffering from diabetes for various time period before pregnancy (diabetes type according to White from B to R) and in a control group (n = 27). Oxysterols (7-ketocholesterol, 7 alpha and 7 alpha-hydroxycholesterols and the sum of 5 alpha, 6 alpha and 5 beta, 6 beta-epoxycholesterols) were determined by thin-layer chromatography with densitometric detection according to the methodology which had been developed in the Chemical Department, the Silesian Medical Academy. The analysis scheme included plasma sample hydrolysis and lipid extraction, oxysterol fraction isolation by solid phase extraction (SPE), separation and identification of individual sterols by TLC technique, and densitometric quantitative analysis of the cholesterol oxidized derivatives mentioned above. We found statistically considerable differences between the concentrations of epoxycholesterol sum and 7-ketocholesterol in both groups, and the concentration was higher in the control group. While analysing the concentrations of the investigated parameters in diabetic pregnant women in II and III trimester we found a statistically considerable increase in oxysterol concentration in III trimester, compared to II trimester. The authors suggest that during complicated diabetic pregnancy the cholesterol oxidation process becomes more intensive, particularly in III trimester, compared to II trimester, both in normal pregnant women and in type I diabetic pregnant women as well.