The objective of this work was to examine the effect of different levels of grazing on muscle nutritional fatty acid (FA) profile, including the beneficial n-3 polyunsaturated fatty acids (PUFA) and cis-9, trans-11 (cis-9, trans-11) 18:2 conjugated linoleic acid (CLA). Thirty male Galician Blond (GB) breed calves were randomly assigned to the following three grazing treatments: (1) continuous pasture grazing for 250 days (P); (2) 197-day grazing followed by a 50-day short period of concentrate-based finishing (PC) and (3) 57-day grazing followed by a 165-day long period of concentrate-based finishing (C). Calves kept sucking their mothers up to the time of slaughter. The slaughter weight was similar for all treatments (about 330 kg). Samples of the longissimus thoracis muscle were used for assessment of chemical composition by near infrared reflectance spectroscopy and FA profiles by gas chromatography. Muscle from C calves was fatter and had higher content in total FA, monounsaturated FA (MUFA), cis-9 18:1 than muscle from P calves, whereas PC muscle had generally intermediate values. No significant treatment difference for total saturated FAs (SFA) was found. Content of potentially beneficial n-3 PUFA (18:3n-3, 20:3n-3, 20:5n-3 and 22:6n-3), cis-9, trans-11 CLA and n-6:n-3 ratio were lower and PUFA : SFA ratio were higher in P than in both C and PC calves. Calves fed exclusively on pasture synthesised higher amounts of beneficial FA than calves finished on concentrate. A 50-day period of concentrate-based finishing was sufficient to offset the synthesis of beneficial FA from pasture grazing.