OBJECTIVE There is no study characterizing the variability of Aggregatibacter actinomycetemcomitans isolates in periodontitis patients in Spain. It is therefore the aim of this investigation to study the serotype distribution of A. actinomycetemcomitans strains isolated from periodontitis patients in Spain. The polymorphism of the genes that codifies the leukotoxin and the operon of the cytolethal-distending toxin (cdt) will also be investigated. METHODS From a total of 701 patients samples, 40 A. actinomycetemcomitans-positive periodontitis patients were included in the study (mean age 45.3, 62.5% females) and their clinical periodontal status was assessed. On average, 1-3 isolates from each patient were sub-cultured and characterized by PCR. RESULTS Using culture the prevalence of A. actinomycetemcomitans was 5.7%. The most frequent serotype was "b", being 30 patients infected by a unique serotype, while 7 patients showed co-colonization, mostly with serotypes "a" and "b". From the 79 pure isolates obtained, 24 were from serotype "a", 30 from serotype "b", 12 from serotype "c" and 4 from serotype "d". Further characterization of these samples showed that none of these 79 isolates demonstrated the 530-bp deletion in the leukotoxin's promoter region that characterizes the JP2 strain. Conversely 65.8% of the isolates were cdt+. CONCLUSIONS The most common serotypes were "a" and "b", being serotype "b" the most prevalent in mono-colonization, while serotypes "e" and "f" were not detected. In the majority of samples, operon that codifies the cdt (65.8%) and the genes responsible for the codification of leukotoxin (100%) were found. None of the isolates were JP2 strains.