<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>The frequency of tumor cell dissemination in the peritoneal cavity is critically related to the progression of peritoneal metastases (PM). Recently, flow cytometry (FCM) has been successfully used to detect tumor cells in malignant effusions.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>A total of 143 single cell suspensions derived from ascites or peritoneal lavages from patients with advanced gastric cancer (GC) were stained with monoclonal antibodies to CD45 and to CD326 as well as 4,6‐diamidino‐2‐phenylindole (DAPI) and FVS780. Using FCM, tumor‐leukocyte ratio (TLR) were calculated from CD45(−)CD326(+) tumor cell counts/ CD45(+)CD326(+) leukocyte counts in DAPI (+) FVS780(−) gated area. In 54 patients, the ratios of CD11b(+), CD4(+) and CD8(+) cells in CD45(+) leukocytes were evaluated in parallel.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>TLR of 69 patients with PM were significantly higher than those of 74 without PM (<jats:italic>p</jats:italic> < .001) and log(TLR) showed strong correlation with peritoneal cancer index scores in 51 PM (+) patients (<jats:italic>r</jats:italic> = 0.439). TLR in PM (+) patients also correlated with the ratio of CD11b (+) myeloid cells (<jats:italic>r</jats:italic> = 0.547), and correlated inversely with those of CD4(+) (<jats:italic>r</jats:italic> = −0.490) and CD8(+) T cells (<jats:italic>r</jats:italic> = −0.648). In PM (−) patients who underwent gastrectomy, TLR never exceeded 0.1% in patients with primary GC without serosal involvement (<T4). However, TLR in patients with T4 GC were significantly higher (<jats:italic>p</jats:italic> < .05) and peritoneal recurrence occurred in 6/8 patients whose TLR exceeded 0.1%.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>TLR in peritoneal fluid reflects tumor burden and the immune environment in peritoneal cavity. Multicolor FCM may provide additional information which can be used for the treatment of the patients with PM.</jats:p></jats:sec>