Trypanosomatid parasites are able to use their flagella for attachment to cuticular surfaces within their arthropod hosts. In this study the attachment mechanism of Leishmania promastigotes was investigated using a new and quantifiable in vitro assay system. The results showed that hemidesmosomal flagellar attachment to three different plastic substrates occurred (Melinex, Polyvinyl, Thermanox). Attachment density was increased by scratching the surface of the substrate or by coating with the hydrocarbons n-octacosane and paraffin. Variation in attachment density was observed, depending on the culture medium and the parasite isolate used. All four species examined, L. braziliensis, L. donovani, L. major and L. mexicana, were capable of flagellar attachment in vitro. Collectively, these data indicate that flagellar attachment is mediated by a non-specific hydrophobic interaction in Leishmania species.