Milk contains mammary epithelial cells that are shed into milk during the lactation process. The opportunity to use milk cell RNA preparations to study mammary physiology provides a significant improvement over the use of biopsy samples and allows easy and repetitive sampling without damaging mammary tissue. In order to determine whether milk purified epithelial cells can reveal the variation of mammary gene expression after changes in milking frequencies, mammary biopsies and milk epithelial cells were prepared from unilaterally once daily milked cows. Five Hostein cows producing 15.7 kg of milk in well balanced udder-halves were subjected to unilateral once- and twice-daily milkings for 8 days. On the last day of unilateral milking, milk was collected from both udder-halves to prepare epithelial cells by centrifugation and a specific purification using an anticytokeratin antibody bound to magnetic beads. Mammary biopsies were performed on both udder-halves after milk collection. RNA was extracted from both milk purified epithelial cells and mammary biopsies. A transcriptomic analysis using a 22k bovine oligonucleotide array was performed to compare the transcript profiles between biopsies from once- and twice-daily milked mammary glands. Data were normalized by a global Loess regression and analysed using a mixture model using the Anapuce R package. Statistical significant raw p-values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure which controls the False Discovery Rate. This analysis showed a differential gene expression of 504 transcripts. We obtained 193 and 232 transcripts up and down regulated, respectively, by once-daily milking compared to twice-daily milking. These transcripts were mainly involved in three functional families: « milk synthesis»; « cell dynamics » and « molecular transports ». The two first families are consistent with previous data showing a depletion of mammary synthesis activity and an activation of cell death during once daily milking. However, in order to precise the implication of the third family, more research has to be initiated to investigate the regulations involving molecular transports. Quantitative real-time reverse transcription PCR (RT-qPCR) analysis confirmed the differential expression in mammary biopsies of 22 mRNA, among 29 selected. The expression of these 22 mRNA and 4 stable transcripts was analyzed by RT-qPCR in the milk purified epithelial cell samples. Twelve transcripts were similarly expressed in both milk purified epithelial cells and mammary biopsies. These transcripts were mostly involved in milk synthesis. Four transcripts on 22 were inversely regulated between epithelial cells and mammary biopsies and ten transcripts on 22 did not significantly vary under once daily milking in milk epithelial cells. These differences may result from the fact that the mammary gland contains other types of cells than the epithelial ones. We therefore confirm that milk purified epithelial cells are a valuable source of mammary transcripts to analyze the effect of milking frequency on milk synthesis. However for peculiar transcripts a difference in gene expression is observed between biopsies and milk purified epithelial cells.