Despite evidence that differences in gene expression levels contribute significantly to phenotypic variation across individuals, there has been only limited effort to study gene expression variation in human tissue. To characterize expression variation in the normal human retina, we utilized a custom retinal microarray to analyze 33 normal retinas from 19 donors, aged 29-90 years. Statistical models were designed to separate and quantify biological and technical sources of variation, including age, gender, eye laterality, gene function and age-by-gender interaction. Although the majority of the 9406 genes analyzed showed relatively stable expression levels across different donors (for an average gene the expression level value of 95 out of a 100 individuals fell within a 1.23-fold range), 2.6% of genes showed significant donor-to-donor variation, with a false discovery rate of 10%. The mean expression ratio standard deviation was 0.15+/-0.8, log2, with a range of 0.09-0.99. Genes selectively expressed in photoreceptors showed higher expression variation than other gene classes. Gender, age and other donor-specific factors contributed significantly to the expression variation of multiple genes, and groups of genes with an age- and gender-associated expression pattern were identified. Our findings show that a significant fraction of gene expression variation in the normal human retina is attributable to identifiable biological factors. The greater expression variability of many genes central to retinal function (including photoreceptor-specific genes) may be partially explained by the dynamics of the vision process, and raises the possibility that photoreceptor gene expression levels may contribute to phenotypic diversity across normal adult retinas. In addition, as such diversity may result in different levels of disease susceptibility, exploring its sources may provide insights into the pathogenesis of retinal disease.