Before the Johns Hopkins Hospital and medical school could be opened in 1889, the Johns Hopkins trustees needed a brilliant teaching staff to reflect their lofty aspirations. William Osler, whose star had continued to climb during his five years in Philadelphia, was chosen to be physician-in-chief. He became one of the "Big Four" of Johns Hopkins, along with William Welch (pathology), William Stewart Halsted (surgery), and Howard Kelly (gynaecology).

The medical school opened four years later in 1893, and Osler's hospital rounds and general clinics became legendary among students, one of whom was Harvey Cushing, who became Osler's first biographer. Thanks to Osler's efforts, this was the first school in the United States to introduce clinical clerkship, meaning fourth-year students would be required to serve as doctors on the wards. The medical school also took the controversial step of admitting women as students, a move which Osler supported - though not without private misgivings.

Osler continued his research on infectious diseases, but continued to broaden his sphere of interest. He was becoming known for his work on internal medicine. He was also becoming a vocal proponent of proper sanitation as a means of preventing disease, and promoted a temperate lifestyle as the key to physical well-being. Osler also published widely in this period, and was kept a busy schedule of public speaking engagements as well. It was during the Baltimore years that Osler wrote the first edition of the classic medical textbook, The Principles and Practice of Medicine, published in 1892, which went through eight editions in Osler's lifetime and sixteen editions in total. The textbook educated generations of doctors and led indirectly to the creation of the Rockefeller Institute of Medical Research. The income from the textbook, combined with his Johns Hopkins salary and the proceeds from his ever-expanding private consulting practice, meant that Osler was financially able to pursue his interest in rare medical texts - the beginning of what would become a huge personal library.

Osler married the widow Grace Revere Gross, a friend from his Philadelphia days, in 1892. Their son, Edward Revere Osler, was born in 1895. The Oslers were fond of entertaining, and often had guests at their home at 1 West Franklin Street, mostly medical students and colleagues.

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