The mammary gland develops in a process known as branching morphogenesis, whereby a distal epithelial bud extends and bifurcates to form an extensive ductal network. Compared with other branched organs, such as the lung and kidney, little is known about the molecular basis of branching in the mammary gland. Here we report a microarray profiling strategy to identify novel genes that may regulate mammary branching. We microdissected terminal end bud (TEB) and mature duct microenvironments from β-actin–green fluorescent protein reporter mice and compared their RNA expression profiles with epithelium-free mammary stroma by means of microarray. We identified 1,074 genes enriched in the TEB microenvironment, 222 genes enriched in the mature duct microenvironment, and 385 genes enriched in both TEB and mature duct microenvironments. The microarray correctly predicted the expression of genes known to be enriched in the epithelium (Ets-5) and stroma (MMP-14) of TEBs and in the mature duct microenvironment (MMP-3). The microarray also correctly predicted the localization of previously uncharacterized genes, such as the TEB-enriched SPRR-1a, the duct-enriched casein-γ, and the general epithelial marker pleiotrophin. Analysis of genes enriched in TEBs revealed several genes in the Wnt (Wnt-2, Wnt-5a, Wnt-7b, Dsh-3, Frizzled-1, Frizzled-2), hedgehog (Dhh), ephrin (Ephrin-B1, Eph-A2), and transcription factor (Twist-1, Twist-2, Snail) families. In situ hybridization verified that these genes were enriched in the TEB epithelium (Wnt-5a, Wnt-7b, Dhh, Eph-A2) or TEB stroma (Wnt-2, Frizzled-1, Ephrin-B1). We discuss the potential roles of these genes in mammary branching morphogenesis. Developmental Dynamics 235:3404–3412, 2006. © 2006 Wiley-Liss, Inc.