David Baltimore was born in New York City. As a high school student, he participated in the Jackson Memorial Laboratory research program in Bar Harbor, Maine. It was his first experience working in a biology research lab and interacting with scientists, and it was the start of his research career.

Baltimore studied biology and chemistry at Swarthmore College. In 1959, the summer of his third year at Swarthmore, Baltimore became one of the first undergraduate research students at Cold Spring Harbor Laboratory. He worked with George Streisinger who introduced him to the "new" field of molecular biology.

After graduating in 1960, Baltimore went to the Massachusetts Institute of Technology to do a Ph.D. in biophysics. He became interested in animal viruses and in 1961 left MIT and went to Rockefeller University to continue graduate work with Richard Franklin. Franklin taught a course on animal viruses that Baltimore had taken in Cold Spring Harbor. Franklin had experimental evidence that showed how certain viruses seem to shut down synthesis of cellular RNA and induce synthesis of viral RNA.

As a post-doctorate, Baltimore continued to study viral systems, specifically viral RNA synthesis. In 1965, Baltimore became a research associate at the Salk Institute where he worked on poliovirus. He found that the RNA genome of poliovirus became the mRNA message once it entered the cytoplasm.

In 1968, Baltimore accepted a position as an associate professor of microbiology at MIT. By this time, he began to suspect that not all RNA viruses replicated in the same manner. Baltimore knew about Howard Temin's DNA provirus hypothesis that viral RNA was a template to make viral DNA, which then became the template for the synthesis of progeny viral RNA. In the '60s, this was a radical idea and a clear departure from the accepted Central Dogma of DNA to RNA to protein. Given his own suspicions, Baltimore thought Temin's theory was logical and was able to prove it by finding the RNA-dependent DNA polymerase, later named reverse transcriptase in RSV and in a mouse tumor virus. Baltimore, Temin and Renato Dulbecco shared the 1975 Nobel Prize in Physiology or Medicine for their discoveries concerning the interaction between tumor viruses and the genetic material of the cell.