The proximities of the three subunits (51, 24, and 9 kDa) of the flavoprotein subcomplex (FP) and five subunits (75, 49, 30, 18, and 13) of the iron-sulfur protein subcomplex (IP) of the bovine NADH: ubiquinone oxidoreductase (complex I) were investigated by cross-linking studies. The cross-linking reagents used were disuccinimidyl tartrate and ethylene glycol bis(succinimidyl succinate). The cross-linked products were identified by sodium dodecyl sulfate gel electrophoresis and immunoblotting with antibodies specific for each subunit. Results showed that the three FP subunits are juxtaposed to one another, and only the 51 kDa subunit of FP is in close proximity to only the 75-kDa subunit of IP. The 75-kDa subunit cross-linked to the 30- and the 13-kDa subunits, the 49-kDa subunit cross-linked to the 30-, 18-, and 13-kDa subunits, and the 30-kDa subunit cross-linked to the 18- and the 13-kDa subunits. No cross-linked products of 75+49-, 75+18-, or 18+13-kDa subunits were detected. These results are consistent with the occurrence of potential electron carriers in FP and IP subunits. These electron carriers are FMN and one iron-sulfur cluster in the 51-kDa subunit, one iron-sulfur cluster in the 24-kDa subunit, and apparently two iron-sulfur clusters in the 75-kDa subunit.