We established a short-term (24 h) culture system for bovine oviduct epithelial cells (BOECs), obtained on day 3.5 of the estrous cycle and evaluated the cells with respect to morphological criteria, marker gene expression, and hormone responsiveness. BOEC sheets were isolated mechanically from the ampulla with similar yields from oviducts ipsi- and contralateral to the ovulation site (57.9 ± 4.6 and 56.4 ± 8.0 × 10 ⁶ cells). BOECs showed > 95% purity and cells cultured for 24 h maintained morphological characteristics present in vivo , as determined by light microscopy, scanning electron microscopy, and transmission electron microscopy. Both secretory cells with numerous secretory granules and ciliated cells with long, well-developed, and vigorously beating kinocilia were visible. Quantitative real-time PCR failed to detect significant differences in transcript levels between ipsi-and contralateral BOECs for the majority of marker genes (estrogen receptors α and β ( ESR1 and ESR2 ), 3-hydroxy-3-methylglutaryl-coenzyme A reductase ( HMGCR ), oviductal glycoprotein 1 ( OVGP1 ), progesterone receptor ( PGR ), and tumor rejection antigen 1 ( TRA1 )) throughout the 24 h culture period. However, the combined data of all time points for glutathione peroxidase 4 ( GPX4 ), a gene previously shown to be expressed at higher levels in the ipsilateral oviduct in vivo , also indicated significantly different mRNA levels in vitro . The expression of marker genes remained stable after 6 h cell culture, indicating only a short adaptation period. Western blot analysis confirmed ESR1 and PGR protein expression throughout the culture period. In agreement with cyclic differences in vivo , estradiol-17β stimulation increased PGR transcript abundance in BOECs. Our novel culture system provides functional BOECs in sufficient quantities for holistic transcriptome and proteome studies, e.g. for deciphering early embryo–maternal communication.