Cervical cancer is the third most common type of cancer amongst women, compared to other neoplasms, with a higher potential for prevention, slow evolution, and has a simple and effective screening test in its detection, cure when diagnosed early. The routine examination in Brazil for the prevention of this type of cancer is the Pap smear test. This study aimed to reveal the knowledge and practice regarding the Pap smear test among cytopathologists. A descriptive and exploratory research, a qualitative approach, performed with 21 cytopathologists, by e-mailing a data collection instrument, from March to April 2018. Data were treated using the Hierarchical Descending Classification in IraMuTeQ software and analyzed by the Discourse of the Collective Subject. The construction of the results was carried out based on aspects of the knowledge of the cytopathologists about the Pap smear, related to the importance of the completion of the post-graduation for its accomplishment, and aspects of cytopathologists practice regarding the relevance of performing the early diagnosis of cervical cancer. We conclude on the need to reorganize the activities in the work process of the health professionals involved in the Pap smear.