Predominant immune cells in colorectal cancer (CRC) are CD4+ T-lymphocytes. The hypotheses are that (i) the CD4+ component in CRC is made up of different functional subtypes; T-helper and T-regulatory cells (TREG), (ii) CD4+ cells in CRC are heterogeneous in their expression of homing markers, and (iii) the presence of homing markers can identify cells with a typical functional response. Peripheral blood lymphocytes were obtained by ficoll separation from 38 patients, with matched tumour (TIL) and lymph node lymphocytes extracted using mechanical disaggregation from 35 and 26 patients respectively. The presence of CD4+CD25+ cells as a putative marker for TREG was assessed. CD4+ cells were stained with a panel of 18 homing markers to identify up-regulation in TIL from CRC. Based on homing markers identified, lymphocyte isolates were separated using Flow Assisted Cell Sorting or MACS separation, and the functional ability of these subpopulations assessed using ELISA. TIL from CRC display up-regulation of homing factors CXCR6, CCR5, and CCR6 (p=0.0001, 0.01782 and 0.5346 respectively). Stimulated CD4+CXCR6+TIL produced interferon-gamma,involved in anti-tumour responses. We have confirmed the presence of a population of inhibitory FoxP3+ and CD4+CD25+ TREG. The techniques validated could be used to monitor the effect of future vaccine trials.