Background The use of the autogenous periosteal graft as biological barrier has been proposed for periodontal regeneration. The aim of this study was to evaluate the histometric findings of the subepithelial connective tissue graft as barrier in intrabony defects compared to a bioabsorbable membrane. Methods Three-walled intrabony defects were created surgically in the mesial aspect of the right and left maxillary canines in five healthy mongrel dogs. The defects were chronified, and two types of barriers were randomly carried out for guided tissue regeneration in a split-mouth design: the test group with a subepithelial connective tissue graft and the control group with a bioabsorbable membrane. The specimens were processed for histometric analyses of the epithelium (E), connective tissue (CT), newly formed cementum (NC), new bone (NB), and total newly formed tissues (NFT). Results The test side showed smaller mean of NC (3.6±1.2), NB (2.1±0.7), and NFT (7.7±0.8) than the control group (NC 7.3±0.5; NB 5.3±1.3; NFT 10.1±2.2; P<0.05). No statistically significant differences were verified for E (test 3.1±2.0; control 2.8±2.1; P>0.05) and CT (test 2.5±1.1; control 2.0± 0.5; P>0.05) between groupsFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Diagnóstico e Cirurgia, Faculdade de Odontologia de Araraquara, Araraquara, RUA HUMAITÁ Nº 1680, CENTRO, CEP 14801903, SP, BrasilDepartment of Master in Dental Sciences, School of Dentistry, Educational Foundation of Barretos (UNIFEB), Av. Prof. Roberto Frade Monte, 389. Jd. Aeroporto, 14783-226 Barretos, SP, BrazilDepartment of Dentistry II, School of Dentistry, Federal University of Maranhão (UFMA), Maranhão, MA, BrazilUniversidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Diagnóstico e Cirurgia, Faculdade de Odontologia de Araraquara, Araraquara, RUA HUMAITÁ Nº 1680, CENTRO, CEP 14801903, SP, BrasilFAPESP: 2003/12810-5