The present study primarily aimed at investigating the oral microbiota in smokers and non-smokers with established gingivitis and monitoring its composition during experimental gingivitis. Secondly, it aimed at examining whether the composition of the microbiota is associated with different levels of gingival inflammation during this experimental gingivitis trial. For this purpose, 25 non-dental university students with gingivitis were recruited. 11 subjects were smokers and 14 were non-smokers. After achieving gingival health, they entered a 14-day experimental gingivitis trial. Plaque and bleeding were assessed before entering into the study (intake), at day 0, day 5 and at day 14 of the experiment. Microbiological samples from mucosal sites and dental plaque (taken at intake, day 0, and day 14) were analysed for the presence of Actinomyces species, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Fusobacterium nucleatum, Peptostreptococcus micros, Porphyromonas gingivalis, Prevotella intermedia and Streptococcus species. At day 14 of the experimental period, the level of plaque formation was not different between smokers and non-smokers, but bleeding scores were lower in smokers than in non-smokers (15% and 30% respectively, p=0.01). The change from natural gingivitis to a state of gingival health and a subsequent change from gingival health to experimentally induced gingivitis was accompanied by quantitative alterations in the cultivable microbiota in both groups. Changes were most prominent in the transition from gingival health to experimental gingivitis and were found in dental plaque for Actinomyces species, C. rectus, F. nucleatum, and P. intermedia. Within the group of non-smokers, a distinction was made between subjects with a 'weak' or 'strong' inflammatory response. No relationship with a single bacterial species could be established which would likely explain the differences in levels of inflammation. It is concluded that differences in response to experimental gingivitis are not caused by major differences in the composition of the oral microbiota.