The close association between restorations with overhanging margins and chronic destructive periodontitis has been known for many years. However, the mechanisms by which overhanging restorations will interact in the pathogenesis of periodontal disease are still unknown. Generally it is accepted that overhanging restorations contribute to the promotion of the disease process by virtue of their capacity to retain bacterial plaque. The purpose of the present study was to determine if the placement of subgingival restorations with overhanging margins results in changes in the subgingival microflora. 9 dental students with clean teeth and clinically healthy gingivae (GI less than 0.1) gave their consent to participate in the study. 5 MOD cast gold onlays with 1 mm proximal overhanging margins were placed in mandibular molars for 19-27 weeks. They were replaced in a cross-over design by 5 similar onlays with clinically perfect margins which served as controls. Another 5 onlays were placed in reverse order in the remaining patients. Prior to and every 2-3 weeks after insertion, subgingival microbiological samples were obtained by inserting a fine sterile paper point for 30 sec into the gingival sulcus subjacent to the restoration. The predominant cultivable flora was determined using continuous anaerobic culturing techniques. Following the placement of restorations with overhanging margins, a subgingival flora was detected which closely resembled that of chronic periodontitis. Increased proportions of Gram-negative anaerobic bacteria, black-pigmented Bacteroides and an increased anaerobe: facultative ratio were noted. Following the placement of the restorations with clinically perfect margins, a microflora characteristic for gingival health or initial gingivitis was observed. Black-pigmented Bacteroides were detected in very low proportions (1.6-3.8%). These changes in the subgingival microflora were obvious irrespective of whether the restorations with the overhanging margins were placed in the first period of the experiment or following the cross-over. Clinically, increasing gingival indices were detected at the sites where overhanging margins were placed. Bleeding on gentle probing always preceded the peak level of black-pigmented Bacteroides. Loss of attachment was not detected in any site. Changes in the subgingival microflora after the placement of restorations with overhanging margins document a potential mechanism for the initiation of periodontal disease associated with iatrogenic factors.