Kitajima, who won the breaststroke double at Athens in 2004 and Beijing four years later, qualified sixth fastest overall when he finished fourth in his semi-final in a time of 59.69 seconds.
South Africa's Cameron van der Burgh set the fastest time of 58.83, just ahead of Italy's Fabio Scozzoli and Australia's Brenton Rickard.
The 29-year-old Kitajima was aided by the slower pace in the second semi-final, with only the top two finishers in that race swimming fast enough to make the final.
Kitajima and Michael Phelps are locked in a race to become the first man to complete a three-peat but with Phelps failing to get on the podium in Saturday's 400 individual medley final, Kitajima has the chance to do it in Sunday's breaststroke final.
The event, however, was tinged with sadness because of the death of world champion Alexander Dale Oen in April.
Norway's Dale Oen, who won the world title in Shanghai last year three days after a bombing and shooting attack in Oslo that killed 77 people, had been at a training camp in Arizona when he collapsed and died.
A medical examiner ruled the 26-year-old had died of severe coronary artery disease and his compatriot Sara Nordenstam said his absence was felt in their team and at the pool.
Dana Vollmer of the United States cemented her place as the favourite to win the women's 100 metres butterfly gold medal after setting the fastest time in the semi-finals.
The reigning world champion broke Inge de Bruijn's 12-year-old Olympic record in the heats then booked her place in the final when she stopped the clock at 56.36 seconds.
Australia's Alicia Coutts was second overall despite swimming just before the final of the 4x100m freestyle relay where she won gold while Swedish world record holder Sarah Sjostrom qualified fourth fastest.
Australia's Jessicah Schipper, a bronze medallist in the event in Beijing four years ago, failed to even make the semi-finals, as did Poland's Otylia Jedrzejczak, the 2004 Olympic champion over 200m.