It became an important place on the camino real (royal road) connecting St. Augustine with Apalachee Province, which was centered on the site of present-day Tallahassee, Florida.

Like other Spanish missions in Florida, Santa Fe de Toloca would have been established in or near an existing Timucua village, belonging either to the Potano or the Northern Utina tribe.

[1] As with other Timucua villages that became part of the Spanish mission system in Florida, the Indians of Santa Fe were greatly affected by epidemics, including bubonic plague in 1613–1617, yellow fever in 1649, smallpox in 1653, and measles in 1659.

After a rebellion by the Western Timucua in 1656, the Spanish hanged a number of the Indian leaders, including the village chief of Santa Fe.

As the Indian population closer to St. Augustine declined, the Spanish became increasingly dependent on corn and other agricultural supplies from Apalachee.

Finally, products could be carried by boat from St. Marks around the Florida peninsula to St. Augustine, bypassing all of the camino real, including the Santa Fe mission.

[3] After the Timucua rebellion of 1656, a number of missions were closed, and others were relocated closer to the camino real connecting St. Augustine to Apalachee Province.

Originally thought to be a 17th-century farmstead or hamlet, the site was eventually recognized as a Spanish mission and, based on documentary evidence, identified as Santa Fé de Toloca.

[5] The site of Santa Fe de Toloca was investigated by non-invasive testing, such as metal detectors, soil resistivity and remote sensing, surface collection and excavation.

This structure was smaller than the church buildings typically found at other missions in Florida, but may have been a chapel or the convento (priest's house).

The location is just inside where remote sensing indicates that an old north–south road, the Santa Fe Trail, appears to fork into two parts that pass around either side of the mission.