PURPOSE. To characterize in vivo morphometry and multispectral autofluorescence of the retinal pigment epithelial (RPE) cell mosaic and its relationship to cone cell topography across the macula. METHODS. RPE cell morphometrics were computed in regularly spaced regions of interest (ROIs) from contiguous short-wavelength autofluorescence (SWAF) and photoreceptor reflectance images collected across the macula in one eye of 10 normal participants (23-65 years) by using adaptive optics scanning light ophthalmoscopy (AOSLO). Infrared autofluorescence (IRAF) images of the RPE were collected with AOSLO in seven normal participants (22-65 years), with participant overlap, and compared to SWAF quantitatively and qualitatively. RESULTS. RPE cell statistics could be analyzed in 84% of SWAF ROIs. RPE cell density consistently decreased with eccentricity from the fovea (participant mean +/- SD: 6026 +/- 1590 cells/mm(2) at fovea; 4552 +/- 1370 cells/mm(2) and 3757 +/- 1290 cells/mm(2) at 3.5 mm temporally and nasally, respectively). Mean cone-to-RPE cell ratio decreased rapidly from 16.+/- at the foveal center to < 5 by 1 mm. IRAF revealed cells in six of seven participants, in agreement with SWAF RPE cell size and location. Differences in cell fluorescent structure, contrast, and visibility beneath vasculature were observed between modalities. CONCLUSIONS. Improvements in AOSLO autofluorescence imaging permit efficient visualization of RPE cells with safe light exposures, allowing individual characterization of RPE cell morphometry that is variable between participants. The normative dataset and analysis of RPE cell IRAF and SWAF herein are essential for understanding microscopic characteristics of cell fluorescence and may assist in interpreting disease progression in RPE cells.