The demystification of how 19th-century novelly designed materials became significant elements of modern technological, economic, and cultural life requires a complete understanding of the material dimensions of historical artifacts. The objects frequently described as the earliest manufactured plastic products—the billiard balls made by John Wesley Hyatt and his associates from the late 1860s—are examined closely for the first time and are found to be more complex and functionally more successful than has been described. Modern analytical techniques such as optical microscopy, scanning electron microscope—energy dispersive X-ray spectroscopy, X-ray fluorescence, micro-Fourier transformed infrared, and handheld/micro-Raman spectroscopies were used to reveal the complex composition of the Smithsonian Institution’s “original” 1868 celluloid billiard ball. Comparisons with billiard and pool balls commercialized from the 1880s to the 1960s showed an unexpected consistency in material formulations. All specimens were made of an unprecedented composite material prepared with a mixture of cellulose nitrate, camphor, and ground bone; the source of the bone was identified as cattle by peptide mass fingerprint (ZooMS). Patent specifications and contemporary journal descriptions explained how and when these formulations emerged. Combining the technical analyses of compositions with a careful reading of the historical record and contemporary descriptions reveals the key elements of the first successful efforts to substitute materials to assist the survival of endangered animals.