The Spectra Optia apheresis system is a newer centrifugation‐based device that in comparison with the COBE Spectra includes features that enhance procedure automation and usability. In this FDA‐approved three‐center two‐arm observational study we characterized the performance of the Spectra Optia for collection of MNCs and CD34+ cells from nonmobilized and granulocyte‐colony stimulating factor (G‐CSF) mobilized healthy donors, respectively. There were a total of 15 evaluable subjects in each arm. Key performance indicators included collection efficiency of MNCs/CD34+ cells, product purity and cellular viability. For nonmobilized donors, median MNC collection efficiency, platelet collection efficiency, product hematocrit and granulocyte contamination were 57%, 12%, 4%, and 1.7%, respectively. For mobilized donors, median MNC collection efficiency, CD34+ cell collection efficiency, platelet collection efficiency, product hematocrit and granulocyte contamination were 61%, 77%, 19%, 4%, and 15%, respectively. Average WBC viability in the mobilized products was 99%. There was one severe (grade 3) adverse event related to citrate toxicity. This study demonstrates that the Spectra Optia can be used for safe and efficacious collection of MNCs, and results obtained are in line with expectations on collection efficiency and product characteristics. Adverse events were limited to those that are well documented in the stem‐cell mobilization and leukapheresis process. As of the time of this writing, FDA 510(k) approval for use of the Spectra Optia device for MNC collection was achieved in the US based partly on the results of this study. J. Clin. Apheresis 29:273–280, 2014. © 2014 Wiley Periodicals, Inc.