A method was developed to determine trace concentrations of a range of individual PCB congeners in biological samples (grass, silage, cattle faeces and milk-fat) which were taken from rural or ‘background’ areas of the UK, in order to prepare a mass balance of PCBs in grazing, lactating cows. A simple milk-fat extraction method was compared to Soxhlet extraction and to whole milk extraction. Results indicated that simply boiling milk-fat in hexane with sodium sulphate present gave a comparable extraction of PCBs to other methods. A clean-up method was devised using acid treated silica gel followed by basic alumina chromatography. Grinding frozen grass or silage with sodium sulphate followed by Soxhlet extraction was found to be the most effective method for these matrices, whilst avoiding the potential contamination/loss of PCBs which can be incurred by air, oven or freeze drying. Soxhlet extraction of cattle faeces, after grinding with sodium sulphate, was found to be effective. A rigorous clean-up was devised which involved passing the extracts through silica gel and acid treated silica gel, followed by size exclusion chromatography (gel permeation chromatography, SEC). 13C12 labelled PCBs were used as recovery standards, quantification was performed using GC-MS. A quality control regime and method validation results are presented. The milk analysis method gave within batch mean recoveries of 69 – 96%, and within batch standard deviations between 1 and 10%. The vegetation analysis method gave within batch mean recoveries of 91 – 116%, and within batch standard deviations between 1 and 11%. The batch to batch mean recovery for milk analysis was 90%, with an RSD of 2% for high spikes and 5% for low spikes; for vegetation analysis the batch to batch average recovery was 106%, with an RSD of 14% for high spikes and 11% for low spikes. ΣPCB concentrations (53 congeners) of 3900 ± 790 pg g−1 milk-fat, 1300 ± 420 pg g−1 dry matter (DM) cattle faeces, 630 ± 140 pg g−1 DM silage and 1350 ± 580 pg g−1 DM grass were found during the study.