Macrophages or activated microglia in the subretinal space are considered a hallmark of some retinal pathologies. We investigated the effects of age, pigmentation and CX₃CR1 deficiency on the accumulation of macrophages/activated microglia in the outer retina of young and old Cx₃cr1^gfp/gfp (CX₃CR1-deficient) or Cx₃cr1^gfp/+ mice on either a pigmented (C57BL/6) or albino (BALB/c) background. Quantitative analysis of immunostained retinal-choroidal whole mounts revealed an increase in subretinal macrophage (SRMΦ) numbers in young Cx₃cr1^gfp/gfp mice compared with Cx₃cr1^gfp/+ mice, however the increase was more marked in albino Cx₃cr1^gfp/gfp mice. In aged mice, large numbers of SRMΦ/activated microglia replete with autofluorescent debris were noted in both old pigmented Cx₃cr1^gfp/gfp and Cx₃cr1^gfp/+ mice proving this accumulation was not CX₃CR1-dependent. While CX₃CR1 deficiency leads to an early onset of SRMΦ accumulation, our data reveal that this change occurs in both aged Cx₃cr1^gfp/+ and Cx₃cr1^gfp/gfp pigmented mice in the absence of marked retinal degeneration and is likely a normal response to aging.