The prevalence of infected and infective black flies was estimated by pool screen polymerase chain reaction (PCR) amplification in the three river basins in Ecuador endemic for onchocerciasis. Mass distribution of ivermectin (Mectizan) resulted in dramatic declines in the prevalence of infected and infective flies. In the Rio Santiago river basin, no infections were detected, suggesting that transmission had ceased. The ratio of infected to infective flies in Simulium exiguum was 10-fold lower than the corresponding ratio for Simulium quadrivittatum, suggesting that S. exiguum is a more-competent vector for Onchocerca volvulus than S. quadrivittatum. However, the prevalence of infective flies in the two species was not different, suggesting that S. quadrivittatum may play an important vectorial role where it is the dominant human-biting species. The data demonstrate that pool screen PCR is an efficient way to monitor transmission in areas subject to control, and to certify an area as free of O. volvulus transmission.