The role of amino acids in cholesterol gallstone formation is not known. Therefore, the aim of the study was to determine the amino acid profile in the bile of patients with and without cholecystolithiasis in relation to bile lithogenicity and telocyte numbers within the gallbladder wall. The study included 23 patients with cholecystolithiasis and 12 gallstone-free controls. The levels of free amino acids in the bile were measured, and telocytes were identified and quantified in the gallbladder muscle wall. The mean values of valine, isoleucine, threonine, methionine, phenylalanine, tyrosine, glutamic acid, serine alanine, proline and cystine were significantly higher in the study group than in the controls (pfrom 0.0456 to 0.000005), and the mean value of cystine was significantly lower in patients with gallstone disease than in the controls (p= 0.0033). The relationship between some of the amino acids, namely alanine, glutamic acid, proline, cholesterol saturation index (CSI) and the number of telocytes was significant (r = 0.5374,p= 0.0051; r = 0.5519,p= 0.0036; and r = 0.5231,p= 0.0071, respectively). The present study indicates a potential relationship between the altered amino acid composition of bile and the reduced number of telocytes in the gallbladder muscle wall in cholelithiasis.