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Abnormalities in CD4+CD25+CD127loFoxP3+ regulatory T cells (Tregs) have been implicated in susceptibility to autoimmune disease. To define migratory Treg subsets that may be implicated in the pathogenesis of diseases manifested in different tissues, we designed 11 and 13 colour flow-cytometry panels to measure the expression of a range of chemokine receptors and integrins on Tregs in the peripheral blood (PB) of healthy individuals (n=44) and patients with rheumatoid arthritis (RA) (n=34) or psoriasis (n=44). We showed that CLA, CCR4, CCR5, CCR6, CCR10 and CD62L were expressed on a higher proportion of CD4+CD25+CD127lo Tregs than conventional CD4+ T cells (Tconvs) in healthy adults. A significantly lower proportion of Tregs expressed the gut homing α4 (CD49d) and β7 when compared to Tregs that expressed the skin homing CLA, CCR4 and CCR10. Furthermore, the skin homing receptors, CLA, CCR4 and CCR10, were expressed on a much higher proportion of Tregs than Tconvs. We used flow cytometry to analyse the levels of CXCR3+, CCR4+, CCR5+ and CCR6+ Tregs in the PB of RA patients. RA patients had significantly reduced levels of effector/memory Tregs in their PB. We also found a significantly reduced frequency of effector/memory Tregs expressing each of the chemokine receptors examined in the PB of RA patients. This could indicate a trafficking deficiency, resulting in less Tregs entering the joints and a consequent disruption of immune homeostasis at the site. We examined the levels of β7+, CD49d+, CLA+, CCR4+, CCR10+, CCR5+, CD62L+, CXCR3+ and CCR6+ Tregs in the PB of psoriasis patients. We found Treg frequency to be increased in the PB of untreated psoriasis patients. However, the frequency of effector/memory Tregs expressing CXCR3 and CCR6 was significantly reduced in the PB of these patients. CXCR3+ and CCR6+ Tregs are known to be crucial to regulating Th1 and Th17 type immune responses, which are both involved in the pathogenesis of psoriasis. Therefore a reduction in iv CXCR3 and CCR6 expression on Tregs could indicate a trafficking deficiency resulting in less Treg entry into psoriatic lesions. We used bioinformatic tools to analyse all flow cytometry data obtained from patients and controls. We hope these methods of analysis will facilitate the uptake of flow cytometry in detecting preclinical disease or selecting patients for optimal treatment.