Objectives. To evaluate US state regulations regarding marijuana, tobacco, and alcohol to determine the extent to which states attempt to govern these substances in early care and education (ECE) settings. Methods. Two independent reviewers assessed regulations enacted by December 2016 for all states and the District of Columbia. Reviewers compared regulations to national standards on the possession or use of marijuana, tobacco, and alcohol in ECE settings. Results. Fewer states regulated marijuana than tobacco or alcohol in ECE settings. More states restricted the use of these 3 substances than they restricted the possession of them. Conclusions. Most states address alcohol or tobacco possession or use in their regulations but should consider updating these provisions to align with national standards. Public Health Implications. Updating and strengthening state childcare regulations regarding marijuana, tobacco, and alcohol possession and use may help protect children in ECE settings, where many children spend a substantial portion of time. As more states legalize marijuana, they may consider updating their regulations and including precise language to better protect children from unintended pediatric marijuana exposure or impaired childcare providers. (author abstract)