Microarray technology is one of the most powerful tools for gene expression profiling in animal sciences. The objectives of this study were to determine the effect of vitrification on gene expression in in vitro- and in vivo-derived bovine embryos, and to identify differential mRNA expression patterns between embryos produced by in vivo v. in vitro conditions. Three pools of in vivo- and in vitro-derived blastocyst-stage embryos were used for microarray analysis. Total RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus Bioscience, Mountain View, CA). Bovine ovarian tissue total RNA was used as the reference. Total RNA samples were amplified using an Ovation^® Pico WTA System (NuGEN Technologies, San Carlos, CA). The bovine 16 846-member microarrays spotted with 70-mer oligonucleotides were purchased from the Bovine Genomics Laboratory, University of Missouri. Amplified cDNA samples were labeled with Alexa Fluor 647 and 546 dyes (Molecular Probes, Eugene, OR), respectively. Combined, labeled samples were dried and resuspended in hybridization buffer containing 50% formamide (vol/vol), 5× SSC, and 0.1% sodium dodecyl sulfate (wt/vol). After denaturation and cooling, cDNA was applied onto a microarray slide. Microarrays were hybridized overnight at 42°C. Following hybridization, the slides were washed with different stringency buffers and water. After drying by centrifugation, the arrays were scanned on a GenePix 4000B scanner (Axon Instruments, Union City, CA). GenePix Pro4.1 software was used for griding and analysis of spot intensities. Good-quality spots were analyzed using the GeneSpring 7.3 software (Agilent Technologies, Inc., CA, Santa Clara, CA). The data were normalized per spot and per array by Lowess normalization. When comparing two treatments, the Welch t-test with Benjamini and Hochberg multiple testing correction was performed to determine the differentially expressed genes between embryo groups. Microarray experiments were performed in 3 biological and 2 technical replicates for all embryo samples. Differentially expressed genes between all embryo groups were identified. The DAVID Functional Annotation Tool was used to analyze the genes that were differentially expressed. The DAVID Functional Annotation Tool determined the co-occurrence probability and provided gene-GO term enrichment analysis to highlight the most relevant GO terms associated with a given gene list. Differentially expressed Kyoto Encyclopedia of Genes and Genomes pathways are as follows: Ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis were significantly upregulated in the fresh embryos, whereas sphingolipid and purine metabolism was the upregulated in the vitrified in vitro-derived embryos. Gene expression was very similar between fresh and vitrified in vivo-derived, as opposed to in vitro-derived, embryos.