Abstract In this study, the effect of sire, sex and intramuscular fat content on the intramuscular fatty acid profile, in particular the long chain polyunsaturated fatty acids (PUFA) was investigated. Therefore, pork samples of the Longissimus thoracis were taken from 121 females and castrates that were the progeny of 5 boars. All animals had been fattened on the same diet and were slaughtered at a live weight of approximately 110 kg. Indices for the activities of Δ9, Δ6 and Δ5 desaturase, as well as elongase activity were estimated from ratios of product to precursor fatty acids. Intramuscular fat content was positively related to the total saturated fatty acid proportion ( r = 0.376; p < 0.01) and the total monounsaturated fatty acid proportion ( r = 0.579; p < 0.01), and inversely correlated with the total PUFA proportion ( r = − 0.637; p < 0.01). A significantly higher index for Δ5 and Δ6 desaturase and elongase activity for PUFA metabolism was observed in females compared to castrate males. Sire had a significant effect on the intramuscular fatty acid profile, notably on the total n − 3 PUFA, and on most individual long chain n − 6 and n − 3 PUFA. The cis-9 C18:1/C18:0 index for Δ9 desaturase activity and the cis-11 C18:1/ cis-9 C16:1 elongase activity index, as well as the combined desaturase and elongase enzyme activities in both the n − 6 and n − 3 PUFA chains were significantly influenced by sire.