A key effector cell in the pathophysiology of Peyronie's disease (PD) is the myofibroblast. Whilst the role of extracellular vesicles (EVs) and the secretome has been elucidated for other diseases, little is known about this in PD. This study aims to elucidate the role of EVs in myofibroblast transformation. Human primary fibroblasts were isolated from tunica albuginea (TA) of PD patients. Conditioned media was generated from cells treated with and without Transforming Growth Factor-ß1 (TGF-ß1). 10% Knockout Serum Replacement (KOSR) was used to avoid EV contamination from foetal calf serum (FCS). Conditioned media was concentrated after 72h and subjected to size-exclusion chromatography to isolate EVs and protein fractions. EVs were analysed via Tunable Resistive Pulse Sensing (TRPS) using the qNano Gold (Izon) to determine size, concentration, shape, and charge of the particles. Cells were treated with EVs in presence/absence of TGF-ß1 and myofibroblast transformation was quantified using In-Cell ELISA (ICE). TRPS analysis revealed that the TGF-ß1-treated cells produced more EVs (39.4 × 108 particles/mL) than untreated cells (7.24 × 108 particles/mL). Untreated cells showed a larger percentage population of particles >80nm compared to TGF-ß1-treated cells, which showed the highest concentration of particles at 70nm. Mean diameter was 92nm and 75nm for untreated and TGF-ß1-treated cells, respectively. There was no difference in charge of particles (zeta potential) between the groups. Slight differences in shape of particles could be detected, with the TGF-ß1-treated group showing more small but potentially elongated particles. No particles above 240nm were detected in either group. Preliminary ICE experiments revealed that isolated fractions impacted myofibroblast transformation. This is the first study to characterize the EV populations of fibroblasts and myofibroblasts in PD. Further work needs to be done to investigate the secretome and to identify the contents of these vesicles and their effect on fibroblast/myofibroblast biology in PD. Funded by ESSM (RG19-04)