Angiotensin TI (Ang TI), a vasoactive octapeptide, has been implicated in cardiac growth and the development of hypertrophy and fibrosis secondary in hypertensive disease. These consequences of Ang II imply an effect on the function and morphology of cardiac interstitial cells (fibroblasts). The present investigation was designed to (1) determine whether neonatal heart fibroblasts (NHFs) possess functional Ang II receptors on their plasma membrane and (2) examine the effects of Ang II on NHFs in vitro using three- and two-dimensional (3D and 2D, respectively) cultures. Several analytic techniques were used to test the specific questions of the present study. Since cardiac fibroblast phenotype can be influenced by culture conditions, both 2D and 3D cultures were used in the present investigations. Reverse-transcriptase polymerase chain reaction and radioligand binding analysis were used to test for the presence of Ang II receptors on NHFs. Both revealed that NHFs in 2D culture possess Ang II receptor mRNA and Ang II receptors. When isolated NHFs were cultured in 3D collagen gels and treated with Ang II, gel contraction was stimulated by NHFs. This effect was attenuated by the specific Ang II receptor antagonist [Sar1, Ala8]Ang II. Ang II-stimulated gel contraction was completely inhibited by extracellular matrix receptor (β1-integrin) antibodies (P<.05), supporting previous studies indicating that collagen gel contraction is mediated via the integrins. Immunofluorescent staining was used to test the localization of cell-surface integrins. A more intense staining pattern for β1- integrin in Ang II-treated versus control cells was observed. Immunoprecipitation of β1-integrin revealed more β1-integrin protein on the surface of Ang II-treated cells (P<.05). These results suggest that Ang II mediates collagen gel contraction via Ang II receptors and β1-integrin, indicating a functional relation between Ang II and β1- integrin on cardiac fibroblasts. We conclude that Ang II influences the ability of cardiac fibroblasts to contract 3D collagen gels and the regulation of membrane-bound integrins in both 2D and 3D cultures of NHFs.