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At Rome, he entered the household of Cardinal Alessandro Farnese, seniore, the future Pope Paul III. He became Cardinal Farnese's vicar general in the diocese of Parma. He also became the governor of Farfa Abbey. After Cardinal Farnese became pope in 1534, Guidiccioni served as a datary from 1536 to 1539. He returned to Lucca, where he took an interest in literary and devotional matters.
In 1539, Pope Paul III recalled Guidiccioni to Rome. Guidiccioni was elected bishop of Teramo on December 12, 1539, and one week later, in the consistory of December 19, 1539, the pope made him a cardinal priest. He was not consecrated as a bishop at this time, though he did receive the red hat and the titular church of San Cesareo in Palatio on January 28, 1540.
He was a member of a congregation of three cardinals that approved the establishment of the Society of Jesus; he was a dissenting voice on the congregation, opposing the approval of the establishment of the order, but after the pope issued the papal bull Regimini militantis Ecclesiae approving the order, Cardinal Guidiccioni became a strong supporter. On August 27, 1540, he was named to a commission to study reform of the Roman Rota.
On March 22, 1542, he resigned the government of Teramo because his duties in Rome made him unable to visit it. He served as governor of Rome during this period. In 1542, he became the inquisitor general of the Roman Inquisition. He also served as Grand Penitentiary and Prefect of the Apostolic Signatura. In all, his published legal writings exceed twenty volumes.
On January 5, 1543, the pope named him to a commission to study church reform. On March 17, 1543, Cardinal Guidiccioni and twelve other cardinals accompanied the pope to Bologna. He was one of eight cardinals appointed on May 11, 1543 to a commission to manage the affairs of the upcoming Council of Trent.
He opted for the titular church of Santa Prisca on September 24, 1543. He served as administrator of the see of Chiusi from April 2, 1544 until February 20, 1545. On November 2, 1544, the pope officially made him a member of the upcoming Council of Trent.
Shortly after the start of the council, on May 26, 1546, he was transferred to the see of Lucca. He finally received episcopal consecration from the hands of Cardinal Rodolfo Pio da Carpi in the Sistine Chapel on August 28, 1546. From January 7, 1547 to January 13, 1548, he was Camerlengo of the Sacred College of Cardinals.
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