The purpose of this study was to examine the distribution and mobility of anionic sites on the surface of fetal trophoblast in contact with maternal blood using polycationic ferritin (PCF) as a probe. Pieces of human placental villi were washed to remove maternal blood, and fresh, unfixed tissue was expose to PCF for varying times, concentrations, and temperatures to determine the effects on labeling patterns. The major findings were: (1) anionic sites were localized almost exclusively on the microvillous portion of the trophoblast surface; intermicrovillous regions of the surface, including the coated pits, were generally not labeled with PCF; (2) PCF binding was present as small clusters on the microvilli. This pattern was observed in tissue incubated 5–10 sec at 4°C and 23°C. The size of the cluster was increased with increased incubation time, suggesting some aggregation or patching can occur; (3) following the fomation of patches, the anionic sites showed no evidence of being cleared from the membrane by endocytosis during incubation subsequent to labeling; (4) the binding of PCF to the surface was reduced by pretreatment of the tissue with neuraminidase. Tissue fixed in glutaraldehyde prior to PCF exposure showed both clustered and more dispersed labeling. The results indicate that anionic sites on human trophoblast surface have a non-random distribution and have restricted mobility on the surface. This may be indicative of a segregation of different membrane proteins and functions within different structural regions of the placental cell surface.