ABSTRACT Bacterial conjugation plays a pivotal role in the evolution and adaptation of genome-reduced mycoplasmas. Despite their fast evolution rate, the conjugative properties of these organisms remain largely understudied, particularly in vivo . In the present study, the ruminant pathogen Mycoplasma agalactiae was used as a model organism to document the conjugative properties of mycoplasmas in environments of increasing complexity, from axenic to cell and organotypic culture conditions. Compared to axenic mating conditions, mycoplasma co-cultivation with goat epithelial cells or bovine precision-cut lung slices (PCLS) resulted in enhanced mating frequencies with high rates of M. agalactiae Integrative and Conjugative Element (ICEA) self-dissemination. These results were conditioned by the presence of eukaryotic cells in the culture and influenced by competition between mating partners but were not limited to M. agalactiae , as similar results were observed with Mycoplasma bovis. Mycoplasma conjugation ex vivo was further characterized by analyzing mycoplasma chromosomal transfer (MCT), a newly discovered mechanism of horizontal exchange of chromosomal DNA that generates mosaic genomes. Although closely associated with ICEA transfer, MCT was detected at low rates under cell and organotypic culture conditions suggesting a complex interplay between these two conjugative processes or a poor viability of the MCT progeny. Finally, mating experiments under nutrient-deprived conditions identified nucleotide stress as a potential factor influencing the modulation of mycoplasma conjugation by eukaryotic host cells. In conclusion, these results suggest that horizontal gene transfer in vivo is likely underestimated and provide valuable models to further studying mycoplasma conjugation ex vivo . IMPORTANCE Conjugation is an evolutionary shortcut that bacteria use to exchange genetic information with their neighbors. Despite the fast evolution rate of the genome-reduced mycoplasmas, their conjugative properties remain largely understudied, particularly in vivo . Here we used the ruminant pathogen Mycoplasma agalactiae to study how mycoplasmas conjugate in co-culture with hosts-derived cells and tissues. Interestingly, conjugation was stimulated when mycoplasmas were co-cultured with eukaryotic cells. This was documented by monitoring the self-propagation of a mobile genetic element known as Integrative and Conjugative Element (ICE) and the exchange of chromosomal DNA leading to the formation of mosaic genomes. While ICE transfer was observed at high frequency, only a few mosaic genomes were detected in the presence of eukaryotic cells. Further data point towards nucleotide stress as a possible factor modulating mycoplasma conjugation in cellular environments. These results suggest that mycoplasma-host interactions may stimulate conjugation in vivo .< Réduire