Pythium insidiosum is an important pathogen of mammals' species, including humans. Equine is the main species affected by this oomycete. P. insidiosum requires an aquatic environment to develop its life cycle, and the susceptible hosts are contaminated when they contact the microorganism in swampy areas. The equine pythiosis is characterized by the formation of irregular masses within the cutaneous lesions, called kunkers, which easily detach from the lesion. From these structures, it is possible to isolate P. insidiosum in pure cultures. The present study aimed to reproduce in vitro the life cycle of P. insidiosum from kunkers of equine clinical lesions. Fifteen kunkers from different horses were tested. It was observed that the discharge of zoospores occurred after 24-48 h of incubation at 37 °C in, respectively, 40 and 47 % of the kunkers evaluated. Only two samples showed no development of the asexual cycle of P. insidiosum under the conditions tested. It was possible to demonstrate that kunkers are able to restart the asexual cycle of P. insidiosum. Based on our in vitro results, we highlight the importance of these structures in the epidemiology of the pythiosis, since kunkers can be a potential source of contamination of this oomycete for aquatic environments.