Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease. It is characterized by persistent joint inflammation, resulting in loss of joint function, morbidity and premature mortality. The presence of antibodies against citrullinated proteins is a characteristic feature of RA and up to 70 % of RA patients are anti-citrulline protein antibody (ACPA) positive. ACPA responses have been widely studied andare suggested to be heterogeneous, favoring antibody cross-reactivity to citrullinated proteins. In this study,we examined factors which may influence cross-reactivity between a commercial human anti-citrullinated fibrinogen monoclonal antibody and a citrullinated peptide. Using a citrullinated pro-filaggrin sequence (HQCHQEST-Cit-GRSRGRCGRSGS) as template, cyclic and linear truncated peptide versions were tested for reactivity to the monoclonal antibody. Factors such as structure, peptide length and flanking amino acids were found to have a notable impact on antibody cross-reactivity. The results achieved contribute to the understanding of the interactions between citrullinated peptides and ACPA, which may aid in the development of improved diagnostics of ACPA.