Knowledge of the biochemical composition of the bovine oviduct in the presence of an embryo is lacking. We have recently reported the detection of alterations to the bovine oviduct transcriptome when multiple embryos, but not a single embryo, were present (Maillo et al. 2015 Biol. Reprod. 92, 144). Thus, we hypothesised that the presence of an embryo would modify the concentration of metabolites and energy substrates in the oviducal fluid. Thus, the aim of the present study was to examine how the presence of an embryo affects metabolites and energy substrates within the oviduct. Cross-bred beef heifers were synchronized, and those in standing oestrus were randomly allocated to cyclic, not bred (n = 7), or pregnant, artificially inseminated (n = 11), groups. All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut, separating ampulla and isthmus. Each portion was flushed with 500 μL of PBS, enabling recovery of any oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (pregnant group) were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Samples of flushing medium from the isthmus and ampulla from oviducts ipsilateral to the corpus luteum from 5 cyclic and 5 pregnant animals (8-cell embryos) were used for metabolites and energy substrates analysis, together with PBS as blank control. Amino acid content in the oviducal fluid was determined by high performance liquid chromatography. Glucose, lactate, and pyruvate were quantified using microfluorometric enzyme-linked assays. Statistical analysis was carried out by 2-way ANOVA with a Bonferroni post hoc for multiple comparisons. No differences were found either in the concentration of metabolites or in the energy substrates between cyclic and pregnant heifers. However, significant differences were detected between ampulla and isthmus in cyclic and pregnant heifers, which is in agreement with our previous findings on the transcriptome between isthmus and ampulla (Maillo et al. 2016 Reproduction 152, 37–46). In pregnant and cyclic heifers, glycine and alanine were more abundant in the ampulla compared with the isthmus (P < 0.01). In addition, in pregnant heifers, arginine was less abundant in the ampulla than in the isthmus (P < 0.01). Energy substrates were also similar between pregnant and cyclic heifers. However, lactate was significantly different between ampulla and isthmus, being present in the ampulla of cyclic and pregnant heifers and being absent from the isthmus (P < 0.05). In conclusion, the content of metabolites and energy substrates in the oviduct varies between the ampulla and isthmus independent of the presence of an embryo.