The objective of this study is to assess the relationship between protozoa in spontaneously expectorated sputum samples and a range of clinical and immunological variables. Clinical details including age, gender, smoking status, and use of oral or inhaled steroids were recorded for a cohort of 199 patients whose spontaneously expectorated sputum samples were submitted to a Cytology Laboratory in Spain between January 2005 and December 2006. Slides were scanned for protozoa under light microscopy and scanned for monocytes/small macrophages highlighted by immunocytochemistry (CD68 monoclonal antibody). One hundred ninety-one patients provided adequate sputum samples, of whom 70 had protozoa in their sputum. There was a strong relationship between the presence of protozoa and monocytes/small macrophages identified under light microscopy (P < 0.001). A binary logistic regression model also indicated a relationship between protozoa and both smoking status and steroid use. The diagnoses in those with protozoa included infection (including tuberculosis), chronic obstructive pulmonary disease (COPD), lung fibrosis, asthma, chronic liver disease, immunosuppression, cancer, pancreatic or renal disease, heart failure, and AIDS. The identified association between protozoa and monocytes/small macrophages in sputum suggests an immune response and warrants further investigation to clarify whether or not these organisms have any pathological significance in this wide range of conditions. Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.