Chlamydia trachomatis consists of two biovars, lymphogranuloma venereum (LGV) and trachoma, that differ in their infectivity in vivo and in vitro. Although addition of exogenous heparin or heparan sulfate in vitro effectively inhibits infectivity of both biovars and inhibits LGV biovar attachment to host cells, trachoma biovar attachment was only modestly inhibited (approximately 30%) by exogenous heparin. To dissect the relationship of heparin inhibition of attachment and infectivity, a heparan sulfate lyase (heparitinase) was used to treat organisms and evaluated for changes in attachment and infectivity. In contrast to heparitinase-treated LGV biovar organisms that lose their ability to attach and infect, treatment of trachoma biovar organisms with a concentration of heparitinase sufficient to reduce trachoma biovar infectivity by > 90%, only inhibited attachment to host cells by approximately 40%. Significantly, attachment could be fully restored for heparitinase-treated organisms of both biovars with exogenous heparan sulfate; however, the coating of the trachoma biovar organisms with heparan sulfate rendered the trachoma biovar similar to the phenotype of the LGV biovar by > 90% sensitivity to heparin inhibition of attachment. These data suggest that the LGV biovar used predominantly a heparin-inhibitable mechanism for attaching to host cells, whereas the trachoma biovar used a heparin-independent means in addition to a heparin-dependent mechanism to adhere to host cells. Once attached, the trachoma biovar, nevertheless, relied on the heparin-dependent pathway to enter host cells.