Pigment cells of mammals are characterized by two different developmental origins: cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain, whereas melanocytes arise from the neural crest. The pigmentation gene tyrosinase is expressed in all pigment cells but differentially regulated in melanocytes and RPE. The tyrosinase promoter does not confer strong expression in pigment cells in vivo, while inclusion of a distal regulatory element at position -15 kb is necessary and sufficient to provide strong expression in melanocytes. Nevertheless, the regulatory elements responsible for correct spatial and temporal tyrosinase expression in the RPE remained unidentified so far. In this report, we show that a 186 kb BAC containing the tyrosinase gene provides transgene expression in both RPE and melanocytes indicating the presence of regulatory sequences required for expression in the RPE. A deletion analysis of the BAC was performed demonstrating that a RPE-regulatory element resides between -17 and -75 kb. Using multi-species comparative genomic analysis we identified three conserved sequences within this region. When tested in transgenic mice one of these sequences located at -47 kb targeted expression to the RPE. In addition, deletion of this regulatory element within a tyrosinase::lacZ BAC provided evidence that this sequence is not only sufficient but also required for correct spatial and temporal expression in the RPE. The identification of this novel element demonstrates that tyrosinase gene expression is controlled by separate distal regulatory sequences in melanocytes and RPE.