Erythrocyte invasion by malaria parasites requires multiple protein interactions. Our earlier studies showed that erythrocyte band 3 is an invasion receptor binding Plasmodium falciparum merozoite surface protein I and 9 (MSPI, MSP9) existing as a co-ligand complex. In this study, we have used biochemical approaches to identify the binding sites within MSP1 and MSP9 involved in the co-ligand complex formation. A major MSP9-binding site is located within the 19 kDa C-terminal domain of MSPI (MSP1(19)). Two specific regions of MSP9 defined as Delta 1a and Delta 2 interacted with native MSPI19. The 42 kDa domain of MSPI (MSP1(42)) bearing MSPI 19 in the C-terminus bound directly to both MSP9/Delta 1a and Delta 2. Thus, the regions of MSPI and MSP9 interacting with the erythrocyte band 3 receptor are also responsible for assembling the co-ligand complex. Our evidence suggests a ternary complex is formed between MSPI, MSP9, and band 3 during erythrocyte invasion by P. falciparum. (c) 2005 Elsevier Inc. All rights reserved.