Primary cultures of human retinal pigment epithelium (RPE) requires young human donors with short post-mortem time and no known retinal diseases. The use of an established human RPE cell line, like ARPE-19, would be a welcomed alternative to primary cultures. This cell line retains many of the characteristics of RPE cells, including cell morphology, functional tight junctions and expression of CRALBP and RPE65. This study was conducted in order to investigate integrin alpha5 expression at both the gene and protein level in the ARPE-19 cell line and compare the results with those obtained with primary cultures of RPE cells. The potential use of this cell line as a substitute for primary cultures of RPE cells was also considered. Integrin alpha5 protein was detected on RPE and ARPE-19 cultures at different confluencies by immunofluorescence and immunoprecipitation analyses. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to study alpha5 mRNA levels. Transient transfections were performed in order to compare alpha5 promoter strength in both types of cells. Immunofluorescence studies showed that both primary RPE and ARPE-19 cells yielded similar alpha5 staining patterns at all cell confluencies. Both immunoprecipitation and RT-PCR analyses provided evidence that sub-confluent and confluent RPE and ARPE-19 cells have similar cell surface alpha5 protein and mRNA levels whereas post-confluent cells had a marked decrease in both protein and transcript levels. ARPE-19 cells show a large increase in promoter strength compared to primary cultures. When compared to primary cultures, the cell line exhibited major differences in the way the alpha5 promoter is regulated, even if both cell types are cultured under identical conditions. This study demonstrates that primary cultures of human RPE and ARPE-19 cells show reductions in both the alpha5 protein and the mRNA when cells reach post-confluency. However, major differences have been observed in the strength of the alpha5 promoter between both cell types. We also show that culturing ARPE-19 cells in a different growth medium alters the transcriptional activity directed by the alpha5 promoter.