Epidemiological and animal studies clearly demonstrate that reproductive events such as puberty, pregnancy and parity play a critical role in the determination of breast cancer risk. We have carried out messenger RNA expression profiling using high-density synthetic oligonucleotide microarrays to identify genes that are differentially expressed between various developmental stages of the murine mammary gland. The use of DNA microarrays to study vertebrate development presents unique analytical challenges compared with expression profiling of homogeneous cell populations. These challenges include accounting for the impact of complex changes in the abundance of multiple cell types on gene expression profiles, as well as identifying functionally relevant patterns of gene expression in the absence of detailed prior knowledge either of the developmental system or of the genes expressed. In order to address these challenges, we have developed a general approach that permits the unbiased identification of biologically relevant patterns of gene expression by identifying statistically significant associations between clustered gene expression patterns and functional gene categories. We have tested the applicability of this approach by analyzing the expression of approximately 5,500 genes during 13 stages of murine mammary gland development. Our findings confirm the utility of this method by demonstrating the ready identification of cellular processes and pathways of known importance in mammary development, as well as shifts in the relative abundance of different cell types within the gland. This approach permitted the identification of genetic pathways with previously unsuspected patterns of developmental regulation, including those involved in fatty acid metabolism, angiogenesis and extracellular matrix synthesis. Our results demonstrate the ability of this analytical approach to suggest new hypotheses regarding mammary development and indicate that this approach will be broadly applicable to the study of complex tissues.