Bernard Shitiabayi, the owner of the Fort-Tenan bus, appeared at the Molo law courts on Thursday.
He was with Cleophas Shimanyula, alias Toto, a Sacco member and a key player in the Western Cross Express Company Limited at Molo Law Courts.
The Wednesday morning accident at Fort Ternan on the Londiani-Muhoroni road has left 56 people dead.
Kericho Police commander James Mugera said Shitiabayi and Toto will have to answer queries.
National Transport and Safety Authority said the bus, operating number KBS 029J, is not found in its website.
NTSA boss Francis Meja said the bus was not licensed to operate at night.
Sources from Nairobi's Machakos Bus Station intimated to KTN News that Homeboyz bus plies Nairobi-Western Kenya route at night.
President Uhuru Kenyatta, Deputy President William Ruto, former Prime Minister Raila Odinga and Nairobi Governor Mike Sonko took to Twitter to share their grief with the families.
The leaders also called upon the motorists to be vigilant on the roads and comply with traffic rules.