We studied immunoglobulin variable heavy-chain (IGHV) repertoire and mutational status in 553 patients with chronic lymphocytic leukemia (CLL) from the Mediterranean area to gain insight into the potential pathogenetic role of antigenic stimulation. The most commonly represented IGHV genes mirrored the usage of normal B cells, with the exception of IGHV1-18, IGHV3-30.3, and IGHV4-59 that were underrepresented. The IGHV3-21 gene, frequently expressed in Northern European CLL, was present only in 16 cases (2.9%). Based on HCDR3 cluster analysis, cases using IGHV3-21 could be grouped in 2 subsets of similar frequency. The first one (7 of 16 cases) carried a similar HCDR3 amino acid sequence (common-HCDR3 subset), virtually identical to the Scandinavian IGHV3-21 CLL. These cases used the IGHJ6 gene; 4 of 7 were unmutated; 6 of 7 carried the V_λ2-14 (IGLV3-21) light-chain gene with a similar LCDR3. All expressed CD38 and had a progressive disease. The second subset (9 of 16) was characterized by heterogeneous HCDR3 rearrangements (nonhomogeneous-HCDR3 subset), diverse IGHJ and IGV light-chain gene usage, variable IGHV mutational status (5 of 9 unmutated), variable CD38 expression, and variable clinical course (4 of 9 progressed). The first subset suggests a potential antigenic element rarely encountered in the Mediterranean area, possibly responsible for a negative outcome. The second subset may reflect the physiologic heterogeneity of expression of IGHV3-21 rearrangements in the normal repertoire and is characterized by a variable clinical outcome. (Blood. 2005;105:1678-1685)