Zika virus (ZIKV) has recently caused a large epidemic in the Americas associated with birth defects. Although ZIKV is primarily transmitted by Aedes spp. mosquitoes, ZIKV RNA is detectable in blood and semen of infected individuals for weeks or months, during which time sexual and other modes of transmission are possible. However, viral RNA is usually detectable for longer than infectious virus is present. We determined the frequency of isolation of infectious virus from semen and serum samples prospectively obtained from a cohort of patients in Puerto Rico. We confirmed positive isolation by tissue culture cytopathic effect, increase in virus genome copy equivalents (GCE), immunofluorescence, and quantitation of infected cells by flow cytometry. These criteria confirmed infectious virus in semen from 8 of 97 patients for up to 38 days after initial detection when virus loads are higher than 1.4x106 GCE/mL. Two serum isolates were obtained from 296 patients. These findings can help guide important prevention guidelines for persons that may potentially be infectious and transmit ZIKV sexually.