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Background Cholecystocentesis can be part of the diagnostic workup of hepatobiliary disease in small animals, but literature on cytological evaluation of bile is scant. Objectives To determine the diagnostic utility of cytological assessment of bile aspirates. Animals Fifty-six and 78 client-owned dogs and cats, respectively, with bile collected by cholecystocentesis and submitted to our diagnostic laboratory between 1999 and 2014. Methods Retrospective study describing cytological findings of bile, concurrent bacterial culture results, hematological and serum biochemical data, gallbladder biopsy results, as well as final diagnosis and complications after cholecystocentesis. Results Infectious agents were found in 30% of canine and 22% of feline bile aspirates, and inflammation in 5% and 19% respectively. Presence of microorganisms was more often detected on cytological examination (24%) than by culture (21%). The most common bacterial isolates were Escherichia coli and Enterococcus spp., isolated from 14.8% and 6.7% of cultured samples respectively. Only increased canine pancreatic lipase immunoreactivity concentration ( cPLI) was significantly associated with the presence of microorganisms, inflammatory cells, or both in bile. Clinically relevant complications of cholecystocentesis occurred in 2 dogs. The majority of the animals undergoing cholecystocentesis suffered from hepatic, pancreatic, gastrointestinal disease, or a combination thereof. Conclusions and Clinical Importance Cytological examination of bile is inexpensive and straightforward, and yields diagnostically relevant information that precedes and complements bacterial culture. [ABSTRACT FROM AUTHOR]