The aim of this human intervention study was to evaluate the Δ9-desaturation of trans-11–18:1 (trans-vaccenic acid; tVA) to cis-9,trans-11–18:2 (c9,t11 conjugated linoleic acid; CLA) and of trans-12–18:1 (t12) to cis-9,trans-12–18:2 after a short-term (7d) and a long-term (42d) supplementation period. The conversion rates of both trans-18:1 isomers were estimated by lipid analysis of serum and red blood cell membranes (RBCM). Subjects started with a 2-week adaptation period without supplements. During the 42d intervention period, the diet of the test group was supplemented with 3g/d of tVA and 3g/d of t12. The diet of the control group was supplemented with a control oil. Serum tVA and t12 levels in the test group increased by fivefold and ninefold after 7d, respectively, and by eight- and 12-fold after 42d, respectively, when compared with the adaptation period (p≤0·002). The serum c9,t11CLA levels increased by 1·7- and 2·0-fold after 7d and 42d, respectively (p≤0·001). After 42d, the test group's RBCM c9,t11CLA content was elevated by 20% (p=0·021), whereas in the control group it was decreased by 50% (p=0·002). The conversion rate of tVA was estimated at 24% by serum and 19% by RBCM. No increase in c9,t12–18:2 was observed in the serum and RBCM, and thus no conversion of t12 could be determined. In conclusion, the endogenous conversion of dietary tVA to c9,t11CLA contributes approximately one quarter to the human CLA pool and should be considered when determining the CLA supply.