Old World climbing fern is one of the most invasive plants in natural areas of central and southern Florida. The fern spreads across the landscape by wind-blown spores and invades isolated and undisturbed habitats such as interior portions of the Florida Everglades. Land managers in Florida have reported that multiple herbicide treatments are required to control the fern, which could indicate that herbicides do not translocate throughout the plant in long-established populations. We conducted a greenhouse study to determine the absorption and translocation patterns in Old World climbing fern using the three herbicides most commonly used for management of this plant by land managers in Florida. Using 14C-labeled herbicides, we evaluated absorption and translocation of glyphosate (2.25 kg ai ha−1), metsulfuron (0.10 kg ai ha−1), and triclopyr (1.68 kg ai ha−1) in Old World climbing fern using five different application scenarios (cut-and-spray, basal spray, 25% foliar spray, 50% foliar spray, and 100% foliar spray). Triclopyr was absorbed to the greatest extent (60.3%) of applied radioactive compounds compared to glyphosate (31.2%) and metsulfuron (19.8%). The majority of radioactivity remained in treated leaves for all herbicides with only small percentages of the absorbed radioactivity being detected in other plant parts. All three herbicides translocated acropetally and basipitally to some extent. Radioactivity, for the most part, translocated evenly throughout the plants but the greatest amount of radioactivity derived from triclopyr occurred in rhizomes when the cut-and-spray and basal applications were used. The radioactivity in rhizomes derived from glyphosate was greater in those treated using cut-and-spray. Based on autoradiographs, there was limited horizontal movement of any herbicide in the rhizomes of Old World climbing fern which could explain why resprouts are observed several weeks following treatment.