Application: Taken in account a beneficial effect of follicular fluid exosomes (ffExo) in reproduction biotechnologies in bovine, out study will enlighten potential factors relying their role on oocyte quality. Introduction: A beneficial effect of ffExo supplementation during IVM on oocyte quality and embryo development was reported in several animal species including cattle (Asaadi et al., 2021; Singina et al., 2022). However, this effect was not observed with ffExo extracted from large dominant follicles (da Silveira et al., 2017). We aimed to compare protein and lipid cargo between ffExo preparations from small follicles (SF) and large dominant follicles (LF). Materials and Methods: Follicular fluid (FF) was aspirated from small antral follicles (3–6 mm) and dominant follicles (>8 mm) of 12 slaughtered cows ovaries. Fractions enriched in ffExo were obtained by 4 serial centrifugations followed by ultracentrifugation at 100 000g. Transmission electron microscopy (TEM) was performed on fixed ffExo. Presence of exosome markers was analyzed by Western blot. Peptide/protein and lipid profiles of ffExo samples (n = 24) were acquired by MALDI-TOF mass spectrometer RapifleX Tissuetyper (Bruker Daltonics) in the 200–1 200 m/z range for lipids and 2 000–30 000 m/z range for proteins, in 9 technical replicates each. Spectral processing and statistical analyses were performed using home R software based on MALDIquant & MALDIquantForeign packages. Peaks annotation were obtained from Top-Down proteomics database and Lipidmaps. Results: Exosomes from SF and LF were similar in size (mean diameters 61.4 nm and 59.1 nm, respectively); however, abundance of exosome specific marker CD81 was significantly higher in SF-ffExo preparations (p < 0.05). Peptido-protein MALDI fingerprints of ffExo revealed 261 peaks. Among them, abundances of 6 proteoforms were up-regulated and 11 were down-regulated in LF compared to SF exosomes (p < 0.05, fold change >1.5). 15 out of 17 differential peaks were annotated and contain protein proteolytic fragments or small proteins known involved in cumulus cells functions. Lipid profiles in negative and positive acquisition modes gathered in total 666 m/z features correspondent to different lipid isoforms. 34 features were significantly up-regulated and 222 down-regulated in ffExo from LF compared to SF (Wilcoxon test, p < 0.01, fold change >2). According to annotation, lysophospholipids LPC and LPE were more abundant in LF exosomes, whereas different phosphatidylcholines and sphingomyelins were more abundant in small follicles. Conclusions: Preparations of follicular fluid exosomes from large dominant or small follicles differed more by their lipid composition than by proteins. Significant difference in membrane lipids might explain different affinity of ffExo to target cells including oocyte.