Between 1535 and 1538 he searched in southwestern Venezuela and northern Colombia for “El Dorado,” in the company of Nikolaus Federmann and then with Philipp von Hutten.
Leaving from the town of Rio Hacha, they followed the eastern flank of the cordilla following the existing salt trade route where it crossed the Andes and entered the lands of the Chibcha.
But Speyer animated them with the hope of discovering the riches of the El Dorado, of which the survivors of Ehinger's expedition, Federmann among them, had brought the first reports.
They continued the march to the south, but, when the rainy season set in, the overflow of the rivers impeded progress, and the consequent fevers decimated their ranks.
Speyer persevered for a long time in his search for the El Dorado, until at last his progress was arrested by a mighty river, probably the Orinoco, or its confluent, the Apure, and early in 1539 he returned to Coro empty handed with only 80 ragged and sickly men out of the host he had led forth more than four years before.