Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the "original" clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.