Our objective was to determine the effect of the duration of grazing before slaughter on the fatty acid composition of muscle fat and s.c. adipose tissue (SAT) of beef heifers. Sixty crossbred Charolais heifers (n = 15 per treatment) were assigned randomly to one of four dietary treatments: 45 animals (Treatments 1, 2, and 3, respectively) were housed at the beginning of the experiment, and 15 (Treatment 4) were fed at pasture. Two groups of 15 heifers were moved to pasture 40 d (Treatment 2) and 99 d (Treatment 3) before slaughter, respectively, resulting in preslaughter grazing periods of 0, 40, 99, or 158 d for Treatments 1, 2, 3, and 4, respectively. Before grazing the predominantly perennial ryegrass pasture, animals were housed and offered grass silage ad libitum and 3 kg of concentrate diet (650 g of grass silage/kg of total DMI). After slaughter, the fatty acid profile of the neutral (NL) and polar lipid (PL) fractions of muscle fat from the LM and the total lipids from SAT were analyzed by gas chromatography. Duration of grazing showed a quadratic tendency on mean carcass weight (P = 0.08), but did not affect growth (P = 0.27) or the lipid content (P = 0.13) of the LM. Increasing the duration of grazing led to a linear increase (P < 0.001) in the concentration (on fresh-tissue basis) of CLA in muscle fat (from 11.80 to 17.75 mg/100 g of muscle in NL, and from 0.52 to 0.82 mg/100 of g muscle in PL) and in SAT (from 3.98 to 10.23 mg/g of SAT; P < 0.001), and increased the concentration of C18:1trans-11 in both muscle fat fractions (P < 0.001) and in SAT (P < 0.001). In the total muscle lipids, the polyunsaturated to saturated fatty acid ratio (P:S) increased from 0.12 to 0.15 with increased duration of grazing following a linear (P < 0.05) and cubic pattern (P < 0.05). Increasing the duration of grazing led to a linear decrease in the n-6:n-3 ratio of muscle fat from 2.00 to 1.32 (P < 0.001), and from 2.64 to 1.65 in the SAT lipids (P < 0.001), mainly as a consequence of the increased concentration of C18:3n-3. It is concluded that muscle fat and SAT fatty acid profile was improved from a human health perspective by pasture feeding, and that this improvement depended on the duration of grazing.