The aim of this trial was to evaluate the influence of castration on fatty acid composition of intramuscular fat. Twenty-four bull calves of Mertolenga breed were randomly assigned to two groups: castrates and intact males. Castration was done at weaning (6 to 8 months of age). After weaning, all animals grazed a rye-grass pasture through 1 year and were then placed in a feed-lot and fed a finishing diet. Three animals of each treatment were slaughtered after a period of 0 (pasture only), 50, 100 and 150 days of feed-lot feeding. All the animals were subjected to the same feeding and management regimes. Fatty acid composition of the intramuscular fat was analyzed in samples of muscle Longissimus lumborum taken after 7 days of ageing.After adjustment for equal intramuscular fat, entire males had significant higher values of C17:0, C18:1trans, C18:2n−6, P/S, n−6/n−3 and C18:2n−6/C20:4n−6 ratios and lower values of C16:0 and C18:1cis-9, indicating that castration has an effect on the fatty acid composition of intramuscular fat of Longissimus lumborum.