The objective of this study was to assess respiratory protection programs for aerosol-transmissible diseases in acute care hospitals for conformance with regulatory requirements and public health guidelines. Twenty-eight representative hospitals were selected by size, location, and ownership in Minnesota and Illinois. Interviews were conducted with 363 health care workers and 171 managers from high-risk departments. Written programs from each hospital were reviewed for required elements. Seventy-seven health care workers were observed donning and doffing a FFR. The most serious deficiency in many written programs was failure to identify a program administrator. Most written programs lacked adequate details about medical evaluation, fit-testing, and training and did not include a comprehensive risk assessment for aerosol transmissible diseases; tuberculosis was often the only pathogen addressed. Employees with the highest probability of tuberculosis exposure were most likely to pick a respirator for close contact, but higher levels of respiratory protection were rarely selected for aerosol-generating procedures. Surgical masks were most commonly selected for close contact with droplet disease- or influenza-infected patients; better protection (e.g., respirator) was rarely selected for higher-risk exposures. Most of the observed health care workers had access to a NIOSH-certified N95 FFR, properly positioned the facepiece, and formed the nose clip. The most frequent deficiencies were failure to correctly place straps, perform a user seal check, and remove the respirator using straps.