Abstract: Streptococcus pneumoniae is a devastating global pathogen. Prevalent in sub-Saharan Africa, pneumococcal serotype 1 is atypical in that it is rarely found as a nasopharyngeal coloniser, yet is described as one of the most common causes of invasive pneumococcal disease. Clonal sequence type (ST)-306 and ST615 are representative of the two major serotype 1 lineages A and C, respectively. Here we investigated the virulence properties and haemolytic activities of these 2 clonal types using in vivo mouse models and in vitro assays. A lethal dose of ST615 administered intranasally to mice led to the rapid onset of disease symptoms and resulted in 90% mortality. In contrast, mice exposed to the same infection dose of ST306 or a pneumolysin (Ply)-deficient ST615 failed to develop any disease symptoms. Interestingly, the 2 strains did not differ in their ability to bind the immune complement or to undergo neutrophil-mediated phagocytosis. Upon comparative genomic analysis, we found higher within-ST sequence diversity in ST615 compared with ST306 and determined that ZmpA, ZmpD proteins, and IgA protease, were uniquely found in ST615. Using cell fractionation and cell contact-dependent assay, we made the unexpected finding that ST615 harbours the expression of two haemolytic variants of Ply: a cell-wall restricted fully haemolytic Ply, and a cytosolic pool of Ply void of any detectable haemolytic activity. This is the first time such a phenomenon has been described. We discuss the biological significance of our observation in relation to the aptitude of the pneumococcus for sustaining its human reservoir.