In the rabbit reproductive model, maternal experimentally induced insulin-dependent diabetes mellitus (expIDD) leads to accumulation of lipid droplets in blastocysts. Cholesterol metabolism is a likely candidate to explain such metabolic changes. Therefore, in the present study we analysed maternal and embryonic cholesterol concentrations and expression of related genes in vivo (diabetic model) and in vitro (embryo culture in hyperglycaemic medium). In pregnant expIDD rabbits, the serum composition of lipoprotein subfractions was changed, with a decrease in high-density lipoprotein cholesterol and an increase in very low-density lipoprotein cholesterol; in uterine fluid, total cholesterol concentrations were elevated. Expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), very low-density lipoprotein receptor (VLDLR), sterol regulatory element binding transcription factor 2 (SREBF2), insulin-induced gene-1 (INSIG1) and cholesterol 7 alpha-hydroxylase (CYP7A1) mRNA was decreased in the liver and low-density lipoprotein receptor (LDLR) mRNA expression was decreased in the adipose tissue of diabetic rabbits. In embryos from diabetic rabbits, the mean (+/- s.e.m.) ratio of cholesterol concentrations in trophoblasts to embryoblasts was changed from 1.27 +/- 2.34 (control) to 0.88 +/- 3.85 (expIDD). Rabbit blastocysts expressed HMGCR, LDLR, VLDLR, SREBF2 and INSIG1 but not CYP7A1, without any impairment of expression as a result of maternal diabetes. In vitro hyperglycaemia decreased embryonic HMGCR and SREBF2 transcription in rabbit blastocysts. The findings of the present study show that a diabetic pregnancy leads to distinct changes in maternal cholesterol metabolism with a minor effect on embryo cholesterol metabolism.