Previously, we reported the antigranulocytic activity of Campylobacter rectus media supernatants containing lipopolysaccharide (LPS) and a 104 kDa protein. Here, we monitored the release of protein and LPS through the growth cycle of C. rectus ATCC 33238 and identified the 104 kDa protein as the cytotoxin. LPS in media supernatants was quantitated by a KDO assay; the 104 kDa protein was detected on immunoblots with specific antibody (A104) and quantitated by amino acid analysis of membrane immobilized protein bands. C. rectus cell product release was independent of cell lysis. Over 24 h, the 104 kDa protein was released linearly while LPS was released in two plateaus; both increased in C. rectus culture supernatants 3 h after inoculation achieving maximum concentrations at 21 h of 3.1 micrograms/ml and 14.6 micrograms/ml, respectively. In 2 h, trypan blue viability assays, 37-47 micrograms of 12, 18 and 24 h supernatant protein killed 33-43% of HL-60 cells. Supernatant toxicity was heat sensitive and inhibited by A104. Sequencing the 16 N-terminal amino acids of the cytotoxin distinguished it from described C. rectus proteins. Similarities between epitopes and amino acid compositions of the Actinobacillus actinomycetemcomitans leukotoxin and C. rectus cytotoxin were observed. These data indicate that C. rectus secretes a 104 kDa cytotoxin.