Invasion of erythrocytes by malaria parasites involves multiple receptor-ligand interactions. To elucidate these pathways, we made use of four parasite clones with differing specificities for invasion, erythrocytes that are mutant for either glycophorin A or B, and enzyme modification of the erythrocyte surface with neuraminidase and trypsin. Neuraminidase alone abolishes invasion of two parasite clones (Dd2, FCR3/A2); these invade after trypsin treatment alone. A third clone (7G8) is unable to invade trypsin-treated erythrocytes. The fourth clone (HB3) can invade after either neuraminidase or trypsin treatment. The receptor for invasion of trypsin-treated erythrocytes was explored in two ways: treatment of trypsin-treated normal cells with neuraminidase, and trypsin treatment of glycophorin B-deficient cells. Both treatments eliminated invasion by all clones, indicating that the trypsin-independent pathway uses sialic acid and glycophorin B. To identify parasite proteins involved in the different pathways, erythrocyte binding assays were performed with soluble parasite proteins from each clone. Based on binding assays using erythrocytes that lack glycophorin A, the parasite protein known as EBA-175 appears to bind predominantly to glycophorin A. In contrast, the glycophorin B pathway does not appear to involve EBA-175, as binding of EBA-175 was similarly reduced to trypsin-treated normal and trypsin-treated glycophorin B-deficient erythrocytes. Thus, the glycophorin B-dependent, sialic acid-dependent invasion of trypsin-treated normal erythrocytes uses a different parasite ligand, indicating two or more sialic-dependent pathways for invasion. Clone 7G8, which cannot invade trypsin-treated erythrocytes, may be missing the ligand for invasion via glycophorin B.(ABSTRACT TRUNCATED AT 250 WORDS)