Cell surface antigens used as markers of in vivo differentiation may not be stable on monocytes maintained under different conditions of in vitro culture. Monocytes were isolated from blood by centrifugation over Percoll or by adherence to plastic dishes, and the cells cultured in suspension or as adherent monolayers. Initially, monocytes obtained by both methods were similar in size, morphology, and surface antigen expression detected with the antimonocyte monoclonal antibodies OKM1, FMC17, PHM2, and PHM3. After culture, cells maintained in suspension were predominantly small, whereas those adherent to plastic rapidly increased in size; however, cytochemical staining for nonspecific esterase and acid phosphatase showed increased enzymic activity by monocytes in both systems, possibly reflecting increased cell maturation. The most striking difference was a substantial loss of FMC17 antigen by most monocytes within four hours in suspension culture, as compared with a qualitative and quantitative increase in expression by plastic adherent cells within two hours. These changes occurred even if the cells were first reacted with lipopolysaccharide. Monocytes taken from suspension culture and allowed to adhere to plastic rapidly synthesized the antigen, a process inhibited by cycloheximide, and conversely, cells removed from plastic progressively displayed decreased FMC17 antigen expression when transferred to suspension culture. No functional role in adherence or phagocytosis has been found for the FMC17 antigen. The results suggest that antigen expression may depend as much on the physical state of the cells as on apparent activation or maturation events.