This study was designed to identify and quantify synoviocyte phenotypes enveloping the canine anterior cruciate ligament (ACL) to test the hypothesis that there are at least two synoviocyte phenotypes, each with distinct quantities and topographical distributions. CD18 and HSP25 epitopes were colocalized in the synovium of 10 normal canine ACLs. Sagittal sections were prepared from medial, central, and lateral aspects of each ACL and phenotypes were quantified in the proximal, middle, and distal aspects of each section. Distinct synoviocyte populations stained positive for CD18 (CD18+) or HSP25 (HSP25+), and a small population of cells stained for both epitopes (DS+). The proportion (mean±SEM) of HSP25+ synoviocytes (57% ±7.5%) was significantly greater than the proportion of CD18+ synoviocytes (27% ± 8.2%), which was significantly greater than the proportion of DS+ synoviocytes (16% ± 3.5%). Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analysis, and immunoelectron microscopy confirmed the presence of CD18 and HSP25 epitopes in the canine ACL. Identification and quantification of ACL synoviocytes may serve as the foundation for future studies involving ACL disease or reconstruction.