Renaissance popes and their advisers were not blind to the need for church reforms, but they resisted calls for a general council representing the entire church and feared that any transformation would mean a loss of power, revenue, and prestige. This changed beginning with Pope Paul III (pontificate 1534–1549), when the papal court became the center of the reform movement rather than its chief opponent. Paul III and his successors supported improvements in education for the clergy, the end of simony (the selling of church offices), and stricter control of clerical life.

In 1542, Pope Paul III established the Supreme Sacred Congregation of the Roman and Universal Inquisition, often called the Holy Office, with jurisdiction over the Roman Inquisition, a powerful instrument of the Catholic Reformation. The Roman Inquisition was a committee of six cardinals with judicial authority over all Catholics and the power to arrest, imprison, and execute suspected heretics. The Holy Office published the Index of Prohibited Books, a catalogue of forbidden reading that included works by Christian humanists such as Erasmus as well as by Protestants. Within the Papal States, the Inquisition effectively destroyed heresy, but outside the papal territories, its influence was slight.

Pope Paul III also called a general council, which met intermittently from 1545 to 1563 at Trent, an imperial city close to Italy. The decrees of the Council of Trent laid a solid basis for the spiritual renewal of the Catholic Church. It gave equal validity to the Scriptures and to tradition as sources of religious truth and authority. It reaffirmed the seven sacraments and the traditional Catholic teaching on transubstantiation. It tackled the disciplinary matters that had disillusioned the faithful, requiring bishops to reside in their own dioceses, suppressing pluralism and simony, and forbidding the sale of indulgences. Clerics who kept concubines were to give them up, and bishops were given greater authority. The council required every diocese to establish a seminary for the education and training of the clergy. For the first time, great emphasis was laid on preaching and instructing the laity, especially the uneducated. The doctrinal and disciplinary legislation of Trent served as the basis for Roman Catholic faith, organization, and practice through the middle of the twentieth century.