Heparin inhibits P falciparum merozoite invasion at initial contact events.P falciparum schizonts (3D7 isolate) in culture were monitored over time for schizont rupture (A) and formation of ring-stage (B) parasites in the presence of heparin (100 μg/mL) or PBS control. Data are mean (range) of one representative assay (2 assays were performed in duplicate). (A) There was a small, but significant, delay of schizont rupture in the presence of heparin *P = .03 for difference in schizont parasitemia for heparin versus control at 15 hours (Mann-Whitney U test). (B) Formation of new ring-stage parasites was greatly inhibited in the presence of heparin, showing that heparin acts predominately by inhibition of merozoite invasion. (C-D) Real-time microscopy of merozoite invasion. (C) Merozoite invasion (arrow) in PBS control cultures demonstrated: (1) initial contact of merozoite with erythrocyte, (2) reorientation, (3) commencement of invasion, (3-5) mechanical invasion and deformation of the erythrocyte, and (6) complete invasion and erythrocyte reformation. (D) Merozoite invasion with cultures containing heparin (100 μg/mL): (1-5) initial contact of merozoite with erythrocyte without reorientation, (6) initial contact is not sustained and merozoite detached. No invasion, reorientation, or erythrocyte deformation was observed in the presence of heparin. Time in seconds is indicated in the bottom right corner. Pictures shown are representative selections from real-time microscopy recordings using the 3D7 P falciparum line. Videos can be found in supplemental data. Microscopy was performed on a Zeiss Axiovert 200M microscope with 63×/1.4 oil (magnification/numeric aperture) objective lens, at humidified tissue culture conditions (5% CO₂, 5% O₂, 90% N₂), 37°C, standard culture media. Images were collected by Zeiss AxioCam MRm with Zeiss Axio Vision software and processed with ImageJ.