Monocytes play an important, yet only partly understood, role in HIV-1 pathogenesis. Two main subsets of peripheral blood monocytes have been described; the major subset of monocytes are phenotypically characterized as being CD14hi/CD16-, and a minor subset (5-15% of total monocytes in healthy individuals), which are CD14lo/CD16hi, have been reported to be expanded in HIV-infected individuals. These CD14lo/CD16hi monocytes differ from the majority of monocytes in a number of ways, including the molecules expressed on their surface and how they function. Here we describe a flow-cytometric assay to identify and compare the expression of a representative surface molecule (CCR5) on CD14hi/CD16- and CD14lo/CD16hi monocytes in small volumes of whole blood, and methods to isolate monocyte subsets by both fluorescence-activated cell sorting (FACS) and magnetic bead sorting.