This study aimed at determining the diagnostic accuracy and role of Fine Needle Aspiration Biopsy (FNAB) in the diagnosis of peripheral lymphadenopathy in Calabar. It further aimed at determining the HIV serological status of affected patients as well as the enhancement of the development of skills and experience in aspiration and diagnostic cytology of lymph node in the University of Calabar Teaching Hospital (UCTH). Patients with persistent peripheral lymphadenopathy for up to 3 months who presented to UCTH Calabar or adjourning hospitals were examined to establish clinically significant lymphadenopathy and rule out regional ulceration, as this was one of the exclusion criteria in the study. Patients with tumour and lymphadenopathy without ulceration were also included. Fine needle aspiration biopsy for cytology using 21G needle on 5ml syringe, full blood count, erythrocyte sedimentation rate and Human Immunodeficiency Virus (HIV) screening tests were done to make initial diagnosis. Subsequently, lymph node biopsy and histology was carried out as control to determine the final diagnosis. Findings were analyzed to determine diagnostic accuracy and the usefulness of FNAB in the diagnosis of lymphadenopathy. The result obtained showed an overall FNAB diagnostic sensitivity of 87%, specificity of 97%, false positive rate of 8%, false negative rate of 6%, positive predictive value of 92%, negative predictive value of 94%, and an overall diagnostic accuracy of 93%. Twenty percent of all patients studied were found to be HIV positive while the hospital incidence of HIV infection among all patients screened was 25%. Highest prevalence of HIV infection was found among the subgroup with reactive lymph node hyperplasia and tuberculosis (28%). In the course of this study, a separate cytology laboratory was developed in the hospital. The turn around time for cytological requests improved from three days to one day. Tremendous experience was gained by the author through this study not only in aspiration cytology but more especially in diagnostic lymphadenectomy. The procedure for lymph node biopsy is described including the pitfalls and complications. It was concluded from this study that fine needle aspiration biopsy is of high diagnostic accuracy and appropriate for routine diagnostic assessment of persistent peripheral lymphadenopathy in Calabar. It was also found to be very useful in preoperative lymph node assessment of malignant tumours with peripheral lymphadenopathy. Routine HIV diagnostic test is recommended for this group of patients