BACKGROUND AIMS: There is real and sustained interest in preparing T-regulatory cells from leukapheresis collections for cellular therapy through the use of simple, effective and reliable methods conforming to Good Manufacturing Practice (GMP). We describe a GMP-ready isolation procedure for CD25(+) products with the use of a sterile docking device, pigtail sampling, a laminar flow hood and the CliniMACS system that uses leukapheresis collections made by two apheresis machines. METHODS: We used CD8/CD19 depletion followed by CD25-positive selection. The median number of CD4(+) cells recovered was 72.5 ± 32.6 × 10(6), of which 60.5% ± 17.8% were CD25(+)FOXP3(+) cells. Suppression of autologous CD25(-) cell proliferation by the cryopreserved CD25(+) products was 51.3% ± 4.4%, 49.0% ± 3.7% and 39.0% ± 3.6% at CD25(+):CD25(-) ratios of 1:1, 1:2 and 1:4 (n = 6), respectively, comparable to suppression by fresh CD25(+) products (53% ± 6.2%, 51% ± 3.3% and 39% ± 7.1%). RESULTS: We found Leukapheresis collections by Cobe Spectra contained more lymphocytes and platelets than collections by Spectra Optia apheresis machine (median, 9.2 × 10(9) versus 6.7 × 10(9); P = 0.04) and platelets (median, 610 × 10(9) versus 170 × 10(9); P = 0.04). The frequency of CD4(+)CD25(+)FOXP3(+) was significantly higher in the leukapheresis (4.85%; 95% confidence interval, 1.95% to 5.38%) than in peripheral blood (3.9%; 95% confidence interval, 2.63% to 6.45%) (P = 0.02). The CD8- and CD19-negative depletion step was associated with significant loss of total CD4(+) T cells (P = 0.001). CONCLUSIONS: Results suggest that functional CD25(+) products can be isolated with a GMP-ready method, and good recovery can be obtained with the use of an optimized cryopreservation protocol. These data and methods show the potential, possibilities and future work needed to isolate target cell populations in a reproducible, time-efficient and cost-efficient manner for clinical applications.